Methods and compositions for treating diabetes, and methods for enriching mrna coding for secreted proteins

ABSTRACT

A previously uncharacterized gene and gene product are disclosed herein that increase blood glucose clearance independent of insulin. Also described is a methodology for enriching for mRNAs transcribing excreted and membrane bound proteins as well as a non-human animal expressing a labeled SEC61b protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/734,981, filed on Sep. 21, 2018. The entire teachings of theabove-identified application are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.2014182349 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

According to the World Health Organization more than 422 million peoplein the world have diabetes. The Endocrine Society is estimating that by2021 the cost burden of diabetes in the United States alone will be $512billion. Current strategies to treat diabetics include insulininjection, augmenting endogenous insulin secretion, increasing glucoseabsorption, or increasing glucose excretion.

Diabetes is a disease derived from multiple causative factors andcharacterized by elevated levels of plasma glucose (hyperglycemia) inthe fasting state. There are two main forms of diabetes mellitus: (1)insulin-dependent or Type 1 diabetes (a.k.a., Juvenile Diabetes) and (2)non-insulin-dependent or Type II diabetes (a.k.a., NIDDM).

Type 1 diabetes is caused by insulin deficiency resulting from loss ofpancreatic beta cells, typically as a result of autoimmune destructionof the islets of Langerhans. Thus, in patients who suffer from type 1diabetes the amount of insulin produced by the pancreatic islet cells istoo low, resulting in elevated blood glucose levels (hyperglycemia).Patients with type 1 diabetes generally require lifelong insulintreatment, but even with frequent daily injections of insulin it isdifficult to adequately control blood glucose levels.

In type 2 diabetic patients, liver and muscle cells lose their normalability to respond to normal blood insulin levels (insulin resistance),resulting in high blood glucose levels. Additionally, Type II diabeticpatients exhibit impairment of beta cell function and an increase inbeta cell apoptosis, causing a reduction in total beta cell mass overtime. Eventually, the administration of exogenous insulin becomesnecessary in type 2 diabetics.

Conventional methods for treating diabetes have included administrationof fluids and insulin in the case of Type 1 diabetes and administrationof various hypoglycemic agents in Type II diabetes. Unfortunately manyof the known hypoglycemic agents exhibit undesirable side effects andtoxicities. Thus, for both type 1 and type 2 diabetes, there is a needfor new treatment modalities.

SUMMARY OF THE INVENTION

A previously uncharacterized gene and gene product are disclosed hereinthat increase blood glucose clearance. Surprisingly and unexpectedly,this gene product (C1ORF127 gene product) lowers blood glucoseindependent of insulin and does not cause hypoglycemia.

The C1ORF127 gene is predicted to code for a protein of 684 amino acidswith a molecular weight of 73 kDa. It is a highly conserved protein thatonly exists in vertebrates. The predicted sequence lacks canonicalsignals for secretion or membrane insertion. C1ORF127 has an N-terminaldomain of 215 amino acids that is highly conserved in vertebrates.Within this domain are two predicted glycosylation sites, one predictedphosphorylation site, five conserved Cysteine residues, and a putativeendopeptidase cleavage site (pro-hormone convertase 2 like, PC2). Thus,like other glucose regulating hormones, C1ORF127 gene product may beprocessed into a heretofore unidentified smaller protein fragment withglucose-lowering activity. The cysteines may participate in proteinfolding or multimerization.

Identification of the gene product and its activity provide a new methodfor treating diabetes and other diseases involving blood glucoseclearance, as well as a new avenue into understanding the causes andeffects of diseases involving blood glucose clearance. Also describedherein is a methodology for enriching for mRNAs transcribing excretedand membrane bound proteins.

Some aspects of the disclosure are directed to a method of treating orpreventing a disorder associated with elevated blood glucose levels in asubject, comprising administering to said subject an effective amount ofan agent that increases the level or activity of a C1ORF127 gene product(herein also sometimes referred to as ERseq08). In some embodiments, theagent increases the level or activity of an endogenous C1ORF127 geneproduct when administered to the subject. In some embodiments, the agentincreases the expression of an endogenous C1ORF127 gene product whenadministered to the subject. In some embodiments, the agent increasesthe secretion of an endogenous C1ORF127 gene product when administeredto the subject. In some embodiments, the agent is a C1ORF127 geneproduct agonist.

In some embodiments, the agent comprises a small molecule, a protein, ora nucleic acid. In some embodiments, the agent comprises a C1ORF127 geneproduct having at least one different post-translational modificationthan a native C1ORF127 gene product. In some embodiments, the agentcomprises a C1ORF127 gene product having at least one substituted,deleted, or added amino acid than a native C1ORF127 gene product. Insome embodiments, the agent comprises a C1ORF127 gene product having adifferent activity or activity level than a native C1ORF127 geneproduct. In some embodiments, the agent comprises a functional portionof a C1ORF127 gene product. In some embodiments, the agent comprises aC1ORF127 gene product comprising a furin cleavage site. In someembodiments, the agent comprises a C1ORF127 gene product without a PC2cleavage site. In some embodiments, the agent comprises a C1ORF127 geneproduct with a furin cleavage site and without a PC2 cleavage site.

In some embodiments, the agent further comprises a pharmaceuticallyacceptable carrier. In some embodiments, the method further comprisesadministration of an additional anti-diabetic therapeutic.

In some embodiments, the agent is a cell expressing C1ORF127 geneproduct. In some embodiments, the cell is an islet cell or a beta-cell.In some embodiments, the cell is autologous to the subject requiringtreatment. In some embodiments, the cell is stem cell-derived. In someembodiments, the stem cell-derived cell is a stem cell-derived betacell. In some embodiments, the cell is encased in a microcapsule orsemi-permeable membrane.

In some embodiments, the agent improves blood glucose clearance whenadministered to the subject. In some embodiments, the blood glucoseclearance property of the agent is independent of insulin activity. Insome embodiments, the agent does not cause hypoglycemia whenadministered to the subject. In some embodiments, the agent has glucosesensitizer activity when administered to the subject. In someembodiments, the agent increases the rate of glucose turnover whenadministered to the subject. In some embodiments, the agent increasesglycolysis when administered to the subject. In some embodiments, theagent increases the rate of glycogen synthesis when administered to thesubject. In some embodiments, the agent has glucagon-like activity whenadministered to the subject.

In some embodiments, the subject has diabetes. In some embodiments, thesubject is human or murine.

In some embodiments, the agent comprises a C1ORF127 protein of SEQ IDNO: 2 or a functional portion or functional variant thereof. In someembodiments, the agent comprises a nucleic acid molecule coding for aC1ORF127 gene product, a functional portion or functional variantthereof, and wherein the nucleic acid comprises the sequence of SEQ IDNO: 1 or a portion thereof. In some embodiments, the agent comprises anucleic acid coding for a C1ORF127 gene product, a functional portion orfunctional variant thereof, and the nucleic acid comprises a sequencehaving at least 90% homology to SEQ ID NO: 1 or a portion thereof.

In some embodiments, administration of the agent corrects a geneticdefect in the subject causing aberrant expression or activity of theC1ORF127 gene product.

Some aspects of the disclosure are directed to an agent that increasesthe level or activity of a C1ORF127 gene product when administered tothe subject.

In some embodiments, the agent increases the level or activity of anendogenous C1ORF127 gene product when administered to the subject. Insome embodiments, the agent increases the expression of an endogenousC1ORF127 gene product when administered to the subject. In someembodiments, the agent increases the secretion of an endogenous C1ORF127gene product when administered to the subject.

In some embodiments, the agent comprises a small molecule, a protein, ora nucleic acid. In some embodiments, the agent comprises a C1ORF127 geneproduct having at least one different post-translational modificationthan a native C1ORF127 gene product. In some embodiments, the agentcomprises a C1ORF127 gene product having at least one substituted,deleted, or added amino acid than a native C1ORF127 gene product. Insome embodiments, the agent comprises a C1ORF127 gene product having adifferent activity or activity level than a native C1ORF127 geneproduct. In some embodiments, the agent comprises a functional portionof a C1ORF127 gene product.

In some embodiments, the agent further comprises a pharmaceuticallyacceptable carrier. In some embodiments, the method further comprisesadministration of an additional anti-diabetic therapeutic.

In some embodiments, the agent improves blood glucose clearance whenadministered to the subject. In some embodiments, the blood glucoseclearance property of the agent is independent of insulin activity. Insome embodiments, the agent does not cause hypoglycemia whenadministered to the subject. In some embodiments, the agent has glucosesensitizer activity when administered to the subject. In someembodiments, the agent increases the rate of glucose turnover whenadministered to the subject. In some embodiments, the agent increasesglycolysis when administered to the subject. In some embodiments, theagent increases the rate of glycogen synthesis when administered to thesubject. In some embodiments, the agent has glucagon-like activity whenadministered to the subject.

In some embodiments, the subject has diabetes. In some embodiments, thesubject is human or murine.

In some embodiments, the agent comprises a C1ORF127 protein of SEQ IDNO: 2 or a functional portion or functional variant thereof. In someembodiments, the agent comprises a nucleic acid coding for a C1ORF127gene product, a functional portion or functional variant thereof, andwherein the nucleic acid comprises the sequence of SEQ ID NO: 1 or aportion thereof. In some embodiments, the agent comprises a nucleic acidcoding for a C1ORF127 gene product, a functional portion or functionalvariant thereof, and the nucleic acid comprises a sequence having atleast 90% homology to SEQ ID NO: 1 or a portion thereof.

Some aspects of the disclosure are related to a method of diagnosing aC1ORF127-related disorder or an increased risk for developing aC1ORF127-related disorder in a test individual, comprising determining aC1ORF127 gene product level in a sample obtained from said testindividual, wherein a C1ORF127 gene product level that is increased ordecreased in said test individual compared to a C1ORF127 gene productlevel in a normal individual is indicative of a C1ORF127-relateddisorder. In some embodiments, the C1ORF127 gene product level isdetected in a blood sample. In some embodiments, the C1ORF127 geneproduct is detected with an antibody. In some embodiments, theC1ORF127-related disorder is diabetes.

Some aspects of the disclosure are related to a method of diagnosing aC1ORF127-related disorder or an increased risk for developing aC1ORF127-related disorder in a test individual, comprising screening thetest individual for a mutation in C1ORF127. In some embodiments, theC1ORF127-related disorder is diabetes.

Some aspects of the disclosure are related to a method of screening fora C1ORF127 gene product receptor agonist, comprising contacting a cellresponsive to a C1ORF127 gene product with a test agent and measuringcell response, wherein if the cell responds then the test agent isidentified as a C1ORF127 gene product receptor agonist. In someembodiments, the cell response is glucose uptake. In some embodiments,the cell is further contacted with an insulin receptor antagonist.

Some aspects of the disclosure are related to a method of enriching formRNAs coding for secreted and membrane bound proteins, comprising: a)providing a cell comprising a Endoplasmic Reticulum (ER) transloconcomprising a label, b) performing sub-cellular fractionalization of thecell and isolating an ER fraction containing the label, and c) isolatingand sequencing mRNA contained in the isolated ER fraction containing thelabel. In some embodiments, the ER translocon component SEC61b comprisesthe label. In some embodiments, the label is a fluorescent label. Insome embodiments, b) comprises contacting the cell with a proteinsynthesis inhibitor, solubilizing the cell plasma membrane, andimmunoprecipitating the ER. In some embodiments, the ER isimmunoprecipitated with an antibody specific for the label. In someembodiments, the protein synthesis inhibitor is cyclohexamide. In someembodiments, the cell plasma membrane (or cell plasma membrane followedby the ER membrane) is solubilized with step-wise concentrations ofdetergent. In some embodiments, the detergent is digitonin, DDM, orboth. In some embodiments, the mRNA is sequenced by next generationsequencing.

In some embodiments, the cell is a beta-cell. In some embodiments, thecell is an induced stem cell or is differentiated from an induced stemcell. In some embodiments, the cell is a diseased cell or exhibits anaberrant state. In some embodiments, the cell is undergoing a stressresponse when contacted with the protein synthesis inhibitor. In someembodiments, the cell is responding to a stimulus when contacted withthe protein synthesis inhibitor.

In some embodiments, the method further comprises performing the methodof enriching for mRNAs coding for secreted and membrane bound proteinsas described herein on a control cell, and comparing the mRNAs isolatedfrom the cell to the mRNAs isolated from the control cell.

Some aspects of the invention are directed to a non-human animal capableof expressing a labeled SEC61b protein. In some embodiments, expressionof the labeled protein is inducible. In some embodiments, the labeledprotein has Cre-dependent expression. In some embodiments, the non-humananimal has inducible expression of the labeled SEC16b protein inbeta-cells. The label is not limited and may be any suitable label inthe art. In some embodiments, the label is a fluorescent protein. Insome embodiments, the label is Green Fluorescent Protein (GFP). In someembodiments, the non-human animal is a mouse or rat. In someembodiments, the non-human animal is a model of diabetes (e.g., NODmodel of type 1 diabetes, model of type 1 diabetes).

BRIEF DESCRIPTION OF THE DRAWINGS

These and other characteristics of the present invention will be morefully understood by reference to the following detailed description inconjunction with the attached drawings. The patent or application filecontains at least one drawing executed in color. Copies of this patentor patent application publication with color drawings will be providedby the Office upon request and payment of the necessary fee.

FIGS. 1A-1B show that C1ORF127 improves glucose clearance independent ofinsulin action. Glucose Tolerance Tests (GTT—2.0 mg/dl glucose bolus)were performed on the third day after hydrodynamic tail vein injections(HTV). FIG. 1A: GTT at Day 3 after HTV injections in ICR outbred micedemonstrated that C1ORF127 can clear glucose from the circulation fasterthan controls. FIG. 1B: GTT at Day 3 after HTV injections in C57Bl6animals dosed with the insulin receptor antagonist S961. Note theelevated blood glucose levels in B. relative to A. and the potentreduction in blood glucose levels in C1ORF127 treated animals startingat 60 minutes relative to controls. In experiments not shown, thereduction in glucose mediated by C1ORF127 starts as early as 15 minutespost glucose injection.

FIGS. 2A-2C shows generation of hPSCs cell lines expression GFP-SEC61β.FIG. 2A: A GFP tag was engineered into the cytosolic domain of SEC61β tofacilitate the isolation of ribsome/translocon complexes at the ERmembrane. FIG. 2B: Strategy for the TALEN-mediated knock-in ofCAAGS::GFP-SEC61β transgene into the AAVS1 locus and perinuclearexpression of GFP in hPSCs. FIG. 2C: Strategy for the CRISPR-mediatedknock-in of GFP-SEC61β into the last exon of the insulin gene. Bottompanels of FIGS. 2B-2C show GFP expression in SC-β cells differentiatedusing this cell line.

FIGS. 3A-3D show ER-Seq enriches for components of the transloconcomplexes and associated ribosomes. FIG. 3A: Sequential biochemicalfractionation approach for the step-wise isolation of cytosolic, ER andnuclear components. ER fraction was subjected to immunopurification ofribosome/translocon complexes and associated RNA. FIG. 3B: Western blotof Sec61β, GFP and ribosomal protein L13a in multiple subcellularfractions after biochemical fractionation. FIG. 3C: RNA gel andidentification of 18S and 28S ribosomal RNA subunits. FIG. 3D:Proteomics-based identification of proteins that are associated withtranslocon complexes. The Uniprot Accession ID, peptide coverage offull-length protein and number of peptides identified are listed. T:total; Cy: cytosolic fraction; Nu: nuclear fraction; Un: unbound ERfraction after immunoprecipation; IP: immunopurified ER fraction.

FIGS. 4A-4C show selective enrichment of mRNAs encoding for factorsinvolved in ER-related processes in self renewing Human Embryonic stemcells. FIG. 4A: Scatterplot of log-normalized microarray signalintensities of genes defected in IP and unfractionated cell extracts.Each dot represents a gene. Differentially expressed genes are coloredlight grey. Candidate secreted or translocon-associated factors arelabeled and colored red. FIG. 4B: Pie chart of predicted subcellularlocalization of IP-enriched genes. FIG. 4C: Top gene ontology terms ofIP-enriched genes.

FIGS. 5A-5F show ER-seq enriches for mRNAs of secreted factors expressedin SC-β cells. FIG. 5A: Diagram of the transgenic hPSC cell line thatexpresses GFP-SEC610 in insulin-expressing β cells. FIG. 5B: Directeddifferentiation protocol of for the generation of SC-β cells using theINS::GFP-SEC61β cell line. FIG. 5C: Scatterplot of log 10-normalizedexpression of genes detected in IP and total unfractionated RNA.Candidate genes are labeled and colored red. Differentially expressedgenes are colored light grey. FIG. 5D: Pie chart of predictedlocalization of IP-enriched genes (fold change>2 relative to total).FIG. 5E: Log 2 enrichment of genes predicted to be part of the secretomeor cytosolic/nuclear (CytoNuc) relative to total unfractionated RNA.Enrichment of IP expression relative to total RNA is shown. FIG. 5F: Topgene ontology terms of IP-enriched genes.

FIGS. 6A-6G shows stage-specific expression patterns oftranslocon-associated mRNAs. FIG. 6A: Diagram of the directeddifferentiation protocol of SC-β cells and the relevant stages at whicheach of the hPSC cell lines were used. FIG. 6B: Heatmap ofdifferentially expressed genes identified by ER-seq across multiplestages of differentiation. FIG. 6C-FIG. 6D: Gene ontology analysis ofgenes that are preferentially expressed in SC-β cells and top terms forbiological processes (FIG. 6C) and cellular components (FIG. 6D). FIG.6E-FIG. 6F: Heatmap of endocrine-specific (FIG. 6E) and unannotated(FIG. 6F) genes across all stages of differentiation displaying a Rcell-specific expression pattern. FIG. 6G: Heatmap of genes with unknownfunction in SC-beta cells.

FIGS. 7A-7E shows identification of C1ORF127 as a glucose loweringactivity. FIG. 7A: Results from Hydrodynamic tail vein injection screen.FIG. 7B: Animals with fasting blood glucose levels below 55 mg/dl wereremoved from further analysis. FIG. 7C: C1ORF127 (red line) clearsglucose from circulation faster than other activities tested. FIG. 7D:for clarity purposes all other treatments were averaged and displayed ingrey. Insulin HTV injected animals (purple) serve as a positive controland also show a robust glucose lowering activity. FIG. 7E:quantification of the glucose lowering effect shows that the differenceis statistically significant.

FIGS. 8A-8B show C1ORF127 is well conserved among vertebrates. FIG. 8A:Protein alignments reveal the high degree of conservation of thisprotein during evolution. FIG. 8B: alignment of human C1ORF127 with itsmouse orthologue, Gm572. Red bar, peptide region used to generate aC1ORF127 antibody used for immunofluorescence analysis and western blot(commercially available antibody). C1ORF127 is a highly conserved 73 kDaprotein lacking a signal peptide. C1ORF127 is expressed in SC-beta cellsand developing mouse pancreas from the inception of endocrine lineage.C1ORF127 is also expressed in adult human and mouse beta-cells.

FIG. 9A-9B show C1ORF127 gene expression. FIG. 9A: in cadaveric humanislets, C1ORF127 is expressed predominantly in beta-cells. FIG. 9B: inSC-beta C1ORF127 is predominantly expressed by SC-beta cells at laterstages of differentiation.

FIGS. 10A-10B show C1ORF127 gene expression. FIG. 10A: t-SNE projectionat late stages of the SC-beta differentiation protocol. C1ORF127 isco-expressed by SC-beta cells, SC-enterochromaffin cells, and inendocrine progenitors. FIG. 10B: the mouse orthologue of C1ORF127, Gm572is expressed by endocrine progenitors.

FIG. 11 shows C1ORF127 gene expression. Analysis of published singlecell RNA seq data from human cadaveric islets demonstrates that C1ORF127is predominantly expressed by beta-cells but it is also expressed bysomatostatin-expressing cells. t-SNE plots.

FIG. 12 shows Immunofluorescence and Western Blot analysis of C1ORF127.Top immunofluorescence, using a commercially available antibody toC1ORF127 it is shown that C1ORF127 is expressed by beta-cells in isletsfrom human cadavers. Note high co-localization with INSULIN staining andabsence from GLUCAGON expressing cells. Bottom, Western blot, Extractsfrom SC-beta cells express C1ORF127 at its predicted molecular weight.

FIGS. 13A-13B show other human tissues expressing C1ORF127. FIG. 13A:Gene expression analysis from the GTEx consortium shows expression inCerebellum and Muscle. FIG. 13B confirms both sites of expression byimmunofluorescence staining. C1ORF127 colocalizes with Laminin in muscleand with Tuj 1 in cerebellum.

FIG. 14 shows S961 model validation. Acute administration of the insulinreceptor antagonist S961 is sufficient to render mice hyperglycemic.Adapted from Schaffer L., et al., Biochemical and Biophysical ResearchCommunications, 2008.

FIG. 15 shows C1ORF127 lowers blood glucose independent of insulinaction. S961 injected 2 hours before, with glucose, and 45 minutes afterglucose injection.. C1ORF127 cleared blood glucose even in the presenceof S961. 1.5 mg/dl glucose bolus.

FIG. 16 shows C1ORF127 improves glucose clearance independent of insulinaction as shown by the effect of ER-seq08 in Streptozotocin (STZ)induced diabetes model. Mice were rendered diabetic by theadministration of STZ (Notice hyperglycemia pre-Hydrodynamic tail veininjection, HTI). Diabetic mice overexpressing C1ORF127 had a markedreduction in blood glucose levels compared to control.

FIG. 17 shows C1ORF127 can lower blood glucose in a high-fat dietobesity model of Type 2 diabetes. DIO—Diet-induced Obesity mice.F-INS—DIO Mice transfected with F-INS—insulin having a Furin cleavagesite. Insulin excreted by the liver.

FIGS. 18A-18C show ER-seq08 improves glucose clearance and does notcause hypoglycemia. FIG. 18A: GTT at Day 3 after HTV injections in ICRoutbred mice demonstrated that ER-seq08 can clear glucose from thecirculation faster than controls. All time points are significantlydifferent. FIG. 18B: Area under the curve measurements from Ademonstrating a significant improvement in glucose clearance inER-seq08-injected mice relative to controls. FIG. 18C: Blood glucoselevels in ER-seq08-injected animals before and after a 16 hour fast,notice there is no significant change in blood glucose levels relativeto controls.

FIGS. 19A-19B show C1ORF127 has primary structure characteristics of apeptide hormone. FIG. 19A: C1ORF127 primary structure with Purple bar:evolutionary conserved domain of unknown function; PC2 site: putativeendopeptidase cleavage site-commonly present in peptide hormones (i.e.INSULIN, GLUCAGON, and AMYLIN); Red bar: A custom rabbit polyclonalantibody generated to the region in red recognizes the full lengthprotein (73 kDa) and a protein of ˜57 kDa. This 57 kDa speciescorrelates with the presumptive PC2 cleavage site. FIG. 19B: is awestern blot: total protein extracts were made from human cadavericislets from non-diabetic (ND) and type 2 diabetic (T2D) patients.Antibody specificity was determined by competition with the peptide usedto immunize the rabbits. Although it is expected that the glucoselowering activity of C1ORF127 resides in the conserved domain of unknownfunction (purple box), the activity may reside on the remainder (57 kDaregion). C1ORF127 is also expressed by delta and gamma cells in theIslets of Langerhans and in muscle and cerebellum. Although, it isspeculated that the glucose lowering activity of C1ORF127 is unique tobeta-cells, other C1ORF127 expression depots may also have glucosemodulating activities.

FIG. 20 shows immunofluorescence staining of adult mouse sections withINSULIN or C1ORF127 antibodies. To our surprise C1ORF127 seems to beexpressed by vesicles not expressing INSULIN. Note the lack of overlapin the 630X inset. At this point we cannot rule out that C1ORF127 mayalso be expressed in some Insulin containing vesicles. This suggeststhat the secretion of C1ORF127 from beta-cells could be modulatedindependently from that of INSULIN and could lead to the discovery of anew class of drugs that exclusively promote C1ORF127 secretion.

FIG. 21 shows C1ORF127 (i.e., ERseq08) is a potent modulator of glucosehomeostasis in beta-cell ablated mice. Streptozotocin (STZ) induceddiabetes is a common mouse model of Type 1 diabetes. All animals arediabetic when fed ad libitum (Fed). After O/N fasting the blood glucoselevels drop. This is followed by an injection of glucose. C1ORF127 isable to clear glucose from the circulation faster than control animals(injected with a red fluorescent protein). Surprisingly, the effect ofC1ORF127 brings animals into the normoglycemic range. These resultssuggest that C1ORF127 is working independent of insulin action. Theresults also suggests C1ORF127 has glucagon-like activity.

FIG. 22 shows Tumors expressing C1ORF127 suppress hyperglycemiaindependent of insulin action. Stably transfected HepG2 cell linesexpressing C1ORF127 (ERseq80) or control fluorescent protein (tdTomato)were implanted under the kidney capsule of Scid/beige mice and tumorswere allowed to grow for a month. A glucose tolerance test was performedafter a four hour fast in the presence of the insulin receptor (INS-R)antagonist S961. Note that the control mice quickly become and stayhyperglycemic for the duration of the experiment. C1ORF127 suppressesthis hyperglycemic excursion.

FIG. 23 shows HEPG2 cells express C1ORF127 protein. A commerciallyavailable C-terminal antibody was used to perform immunoblot analysis onextracts from stably transfected HEPG2 cells expressing C1ORF127 orcontrol. The expected ˜73 kDa product is readily detected.

FIG. 24 shows an immunoblot with a rabbit polyclonal antibody raisedagainst a peptide fragment recognizing C1ORF127 (red bar in schematicabove). A specific protein band of approximately 34 kDa is seen in bothhealthy and type-2 diabetic islets and disappears when the antibody isincubated with blocking peptide. The housekeeping protein Vinculin isshown for comparison. This is consistent with RNA expression data. Type2 diabetic islets appear to have more C1ORF127. Rabbit polyclonalantibody raised against a portion of the conserved domain of C1ORF127located between the N-terminal and PC2 cleavage site.

FIGS. 25A-25C show GreenER mouse reporter. FIG. 25A: construct used formES targeting. mES positive clones were infected with Cre-virus to showexcision of the loxp cassette and marking of the ER. FIG. 25B: tail tipfibroblasts from Green-ER mice crossed to a ubiquitous reporter. Notestereotypical ER-staining. FIG. 25C: beta-cell Green-ER mice demonstratetissue-specific recombination in Insulin producing cells and appropriatesub-cellular localization.

FIG. 26 shows a mouse liver transduced with GFP after HTVi.

FIGS. 27A-27B show C1ORF127 lowers blood glucose in an ablation modelindependently of insulin action. FIG. 27A: Blood glucose levels inC1ORF127 tail vein injected Streptozotocin (STZ) ablated animals. Theseanimals were fasted overnight and administered with glucose, then theirblood glucose level was monitored. This larger cohort showed the sameglucose clearance phenotype as previous. FIG. 27B: Eight weeks later,the same cohort was again tail vein injected with either C1orf127(ERseq08) or tdTomato. Three days later, these mice were fastedovernight, and treated with S961 at 2-hours before glucose injection andat the time of glucose injection. Their blood glucose levels weremonitored. The Streptozotocin ablation, while enough to cause severediabetes in the mice, leaves a small population of insulin-producingbeta-cells. The S961 insulin-antagonist treatment of the ablated animalsshows that residual insulin is not responsible for the glucose clearancephenotype. NOTE: Both experiments utilized a glucometer with a maximalreading of 750 mg/dl, instead of the previous maximum of 600 mg/dl.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the inventions described herein arise from a novel methodof enriching for mRNAs associated with the endoplasmic reticulum andthus likely coding for secreted proteins. When performing this method onbeta-cells, a previously uncharacterized mRNA was isolated. When anucleic acid sequence coding for the gene product of this mRNA (theC1ORF127 gene product) was expressed in mice, the mice had improvedblood glucose clearance independent of insulin. The C1ORF127 geneproduct therefore provides a new methodology for treatment of diseasesand conditions involving blood glucose clearance, such as diabetes.

Methods of Treating or Preventing Disorders Associated with ElevatedBlood Glucose Levels

Some aspects of the disclosure are related to a method of treating orpreventing a disorder associated with elevated blood glucose levels in asubject, comprising administering to said subject an effective amount ofan agent that modulates the level or activity of a C1ORF127 geneproduct.

As used herein a disorder associated with elevated blood glucose levelsis any disorder wherein the subject has elevated blood glucose levels.In some embodiments, the disorder is diabetes (e.g., Type I diabetes orType II diabetes), metabolic syndrome, glucose intolerance, or obesity.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient”, “individual” and“subject” are used interchangeably herein. Preferably, the subject is amammal. The mammal can be a human, non-human primate, mouse, rat, dog,cat, horse, or cow, but are not limited to these examples. Mammals otherthan humans can be advantageously used, for example, as subjects thatrepresent animal models of, for example, diabetes. In addition, themethods described herein can be used to treat domesticated animalsand/or pets. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g., diabetes) or one or more complications related to such acondition, and optionally, but need not have already undergone treatmentfor a condition or the one or more complications related to thecondition. Alternatively, a subject can also be one who has not beenpreviously diagnosed as having a condition in need of treatment or oneor more complications related to such a condition. Rather, a subject caninclude one who exhibits one or more risk factors for a condition or oneor more complications related to a condition. A “subject in need” oftreatment for a particular condition can be a subject having thatcondition, diagnosed as having that condition, or at increased risk ofdeveloping that condition relative to a given reference population.

The term “agent” as used herein means any compound or substance such as,but not limited to, a small molecule, nucleic acid, polypeptide,peptide, drug, ion, etc. An “agent” can be any chemical, entity ormoiety, including without limitation synthetic and naturally-occurringproteinaceous and non-proteinaceous entities. In some embodiments, anagent is nucleic acid, nucleic acid analogues, proteins, antibodies,peptides, aptamers, oligomer of nucleic acids, amino acids, orcarbohydrates including without limitation proteins, oligonucleotides,ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, andmodifications and combinations thereof etc. In some embodiments, theagent is selected from the group consisting of a nucleic acid, a smallmolecule, a polypeptide, and a peptide. In certain embodiments, agentsare small molecule having a chemical moiety. For example, chemicalmoieties included unsubstituted or substituted alkyl, aromatic, orheterocyclyl moieties including macrolides, leptomycins and relatednatural products or analogues thereof. Compounds can be known to have adesired activity and/or property, or can be selected from a library ofdiverse compounds.

“Small molecule” is defined as a molecule with a molecular weight thatis less than 10 kD, typically less than 2 kD, and preferably less than 1kD. Small molecules include, but are not limited to, inorganicmolecules, organic molecules, organic molecules containing an inorganiccomponent, molecules comprising a radioactive atom, synthetic molecules,peptide mimetics, and antibody mimetics. As a therapeutic, a smallmolecule may be more permeable to cells, less susceptible todegradation, and less apt to elicit an immune response than largemolecules.

As used herein, the term “polypeptide” or “protein” is used to designatea series of amino acid residues connected to the other by peptide bondsbetween the alpha-amino and carboxy groups of adjacent residues. Theterm “polypeptide” refers to a polymer of protein amino acids, includingmodified amino acids (e.g., phosphorylated, glycated, glycosylated,etc.) and amino acid analogs, regardless of its size or function. Theterm “peptide” is often used in reference to small polypeptides, butusage of this term in the art overlaps with “protein” or “polypeptide.”Exemplary polypeptides include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, as well as both naturally and non-naturally occurringvariants, fragments, and analogs of the foregoing.

The term “nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The terms“nucleic acid” and “polynucleotide” are used interchangeably herein andshould be understood to include double-stranded polynucleotides,single-stranded (such as sense or antisense) polynucleotides, andpartially double-stranded polynucleotides. A nucleic acid oftencomprises standard nucleotides typically found in naturally occurringDNA or RNA (which can include modifications such as methylatednucleobases), joined by phosphodiester bonds. In some embodiments anucleic acid may comprise one or more non-standard nucleotides, whichmay be naturally occurring or non-naturally occurring (i.e., artificial;not found in nature) in various embodiments and/or may contain amodified sugar or modified backbone linkage. Nucleic acid modifications(e.g., base, sugar, and/or backbone modifications), non-standardnucleotides or nucleosides, etc., such as those known in the art asbeing useful in the context of RNA interference (RNAi), aptamer, CRISPRtechnology, polypeptide production, reprogramming, or antisense-basedmolecules for research or therapeutic purposes may be incorporated invarious embodiments. Such modifications may, for example, increasestability (e.g., by reducing sensitivity to cleavage by nucleases),decrease clearance in vivo, increase cell uptake, or confer otherproperties that improve the translation, potency, efficacy, specificity,or otherwise render the nucleic acid more suitable for an intended use.Various non-limiting examples of nucleic acid modifications aredescribed in, e.g., Deleavey G F, et al., Chemical modification ofsiRNA. Curr. Protoc. Nucleic Acid Chem. 2009; 39:16.3.1-16.3.22; Crooke,ST (ed.) Antisense drug technology: principles, strategies, andapplications, Boca Raton: CRC Press, 2008; Kurreck, J. (ed.) Therapeuticoligonucleotides, RSC biomolecular sciences. Cambridge: Royal Society ofChemistry, 2008; U.S. Pat. Nos. 4,469,863; 5,536,821; 5,541,306;5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308;5,773,601; 5,886,165; 5,929, 226; 5,977,296; 6,140,482; 6,455,308 and/orin PCT application publications WO 00/56746 and WO 01/14398. Differentmodifications may be used in the two strands of a double-strandednucleic acid. A nucleic acid may be modified uniformly or on only aportion thereof and/or may contain multiple different modifications.Where the length of a nucleic acid or nucleic acid region is given interms of a number of nucleotides (nt) it should be understood that thenumber refers to the number of nucleotides in a single-stranded nucleicacid or in each strand of a double-stranded nucleic acid unlessotherwise indicated. An “oligonucleotide” is a relatively short nucleicacid, typically between about 5 and about 100 nt long.

The term “modulates C1ORF127 gene product level or activity” refers toupregulation (activation or increasing activity) or downregulation(inhibition) of a gene product (e.g., C1ORF127 protein) level, activityor function. In one embodiment, the modulation occurs by directlyincreasing or inhibiting the activity of a gene product, i.e., viadirect physical interaction with the gene product. In one embodiment,the activity of the gene product is modulated indirectly, for example,in signaling, by activating or inhibiting an upstream effector of thegene product activity. In some embodiments, the agent increases thelevel or activity of endogenous C1ORF127 gene product when administeredto a subject. In some embodiments, the agent increases the expression ofendogenous C1ORF127 gene product when administered to a subject. In someembodiments, the agent increases the secretion of endogenous C1ORF127gene product when administered to a subject. In some embodiments, theagent is an agonist of endogenous C1ORF127 gene product.

The terms “decrease,” “reduce,” “reduced,” “reduction,” “decrease,” and“inhibit” are all used herein generally to mean a decrease by astatistically significant amount relative to a reference. However, foravoidance of doubt, “reduce,” “reduction” or “decrease” or “inhibit”typically means a decrease by at least 10% as compared to a referencelevel and can include, for example, a decrease by at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, up to andincluding, for example, the complete absence of the given entity orparameter as compared to the reference level, or any decrease between10-99% as compared to the absence of a given treatment.

The terms “increased,” “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%, or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or more as compared to areference level.

As used herein, “treat,” “treatment,” or “treating” when used inreference to a disease, disorder or medical condition, refer totherapeutic treatments for a condition, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a symptom or condition. The term “treating”includes reducing or alleviating at least one adverse effect or symptomof a condition. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a condition is reduced or halted. Thatis, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation or at least slowing of progress orworsening of symptoms that would be expected in the absence oftreatment.

The methods described herein may lead to a reduction in the severity orthe alleviation of one or more symptoms of the disorder. Symptoms ofdiabetes include, for example, elevated fasting blood glucose levels,blood pressure at or above 140/90 mm/Hg; abnormal blood fat levels, suchas high-density lipoproteins (HDL) less than or equal to 35 mg/dL, ortriglycerides greater than or equal to 250 mg/dL (mg/dL=milligrams perdeciliter of blood). Other symptoms of diabetes include for examplefrequent urination, excessive thirst, extreme hunger, unusual weightloss, increased fatigue, irritability, or blurry vision.

In some embodiments, the methods disclosed herein delays the onset ofdiabetes. Delaying the onset of diabetes in a subject refers to delay ofonset of at least one symptom of diabetes, e.g., hyperglycemia,hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness,memory loss, renal failure, cardiovascular disease (including coronaryartery disease, peripheral artery disease, cerebrovascular disease,atherosclerosis, and hypertension), neuropathy, autonomic dysfunction,hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1week, at least 2 weeks, at least 1 month, at least 2 months, at least 6months, at least 1 year, at least 2 years, at least 5 years, at least 10years, at least 20 years, at least 30 years, at least 40 years or more,and can include the entire lifespan of the subject.

As used herein, “prevent” when used in reference to a disease, disorderor medical condition, refers to reducing or eliminating the likelihoodof development of the disease, disorder or medical condition.

As used herein, the term “administering,” refers to the placement of theagent as disclosed herein into a subject by a method or route whichresults in delivery to a site of action. The agent can be administeredby any appropriate route which results in an effective treatment in thesubject. Thus administration via the intravenous route is specificallycontemplated. However, with appropriate formulation, other routes arecontemplated, including, for example, intranasally, intraarterially;intra-coronary arterially; orally, by inhalation, intraperitoneally,intramuscularly, subcutaneously, intracavity, or by other means known bythose skilled in the art. The agents are administered in a mannercompatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered and timing depends onthe subject to be treated, capacity of the subject's system to utilizethe active ingredient, and degree of therapeutic effect desired.

A “therapeutically effective amount” is an amount of an agent that issufficient to produce a statistically significant, measurable change in,for example, blood glucose clearance. Such effective amounts can begauged in clinical trials as well as animal studies. A treatment isconsidered “effective treatment,” as the term is used herein, if any oneor all of the signs or symptoms are improved or ameliorated, e.g., by atleast 10% following treatment with an agent as described herein.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill.

In some embodiments, the agent comprises a small molecule, a protein, ora nucleic acid. In particular aspects, desirable agents (e.g.,compounds) increase levels or activity (e.g., by increasing expressionand/or secretion) of C1ORF127 gene product (e.g., C1ORF127 protein).Suitable compounds/agents include, but are not limited to, chemicalcompounds and mixtures of chemical compounds, e.g., small organic orinorganic molecules; saccharides; oligosaccharides; polysaccharides;biological macromolecules, e.g., peptides, proteins, and peptide analogsand derivatives; peptidomimetics; nucleic acids; nucleic acid analogsand derivatives; extracts made from biological materials such asbacteria, plants, fungi, or animal cells or tissues; naturally occurringor synthetic compositions; peptides; aptamers; and antibodies, orfragments thereof.

A compound/agent can be a nucleic acid RNA or DNA, and can be eithersingle or double stranded. Example nucleic acid compounds include, butare not limited to, a nucleic acid encoding a protein activator orinhibitor (e.g. transcriptional activators or inhibitors),oligonucleotides, nucleic acid analogues (e.g. peptide-nucleic acid(PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA)etc.), antisense molecules, ribozymes, small inhibitory or activatingnucleic acid sequences (e.g., RNAi, shRNAi, siRNA, micro RNAi (mRNAi),antisense oligonucleotides etc.)

A protein and/or peptide agent can be any protein that modulates geneexpression or protein activity. Non-limiting examples include mutatedproteins; therapeutic proteins and truncated proteins, e.g. wherein theprotein is normally absent or expressed at lower levels in the targetcell. Proteins can also be selected from genetically engineeredproteins, peptides, synthetic peptides, recombinant proteins, chimericproteins, antibodies, midibodies, minibodies, triabodies, humanizedproteins, humanized antibodies, chimeric antibodies, modified proteinsand fragments thereof. A compound or agent that increases expression ofa gene or increases the level or activity of a protein encoded by a geneis also known as an activator or activating compound. A compound oragent that decreases expression of a gene or decreases the level oractivity of a protein encoded by a gene is also known as an inhibitor orinhibiting compound. In some embodiments, a protein or polypeptide agentmay be a functional variant or functional fragment of native C1ORF127gene product.

In some embodiments, C1ORF127 has a nucleotide sequence of SEQ ID NO: 1:

ATGGGGTTGGAGCGGAGTGATCGCTACATAATGAAGTGTCCGATGCTAAGGTCAAGGCTGGGTCAGGAAAGCGTCCACTGTGGGCCCATGTTCATCCAGGTCTCCCGGCCCCTGCCCCTGTGGAGGGACAATAGACAGACTCCATGGCTGCTGTCCCTTCGAGGGGAGCTGGTGGCTTCTCTTGAAGACGCCAGCCTGATGGGACTGTATGTGGACATGAATGCCACCACTGTCACCGTCCAAAGCCCGAGACAAGGCCTTCTTCAGAGGTGGGAGGTGCTGAACACCTCTGCTGAGCTCCTGCCACTATGGCTGGTGAGCGGTCACCATGCCTATTCTTTAGAAGCTGCTTGCCCACCGGTGTCATTCCAGCCAGAGTCGGAGGTCTTAGTTCACATCCCCAAGCAGAGACTGGGTCTAGTCAAAAGAGGTTCCTACATTGAGGAAACCCTGAGCCTCAGATTCCTCCGAGTCCACCAGTCCAACATCTTTATGGTGACTGAGAACAAGGACTTTGTGGTGGTCAGCATTCCGGCGGCCGGGGTGCTCCAGGTCCAGCGATGCCAAGAAGTCGGAGGAACCCCGGGAACACAAGCTTTCTATAGGGTAGACCTGAGCCTGGAATTTGCCGAGATGGCTGCCCCGGTCCTCTGGACAGTGGAGAGCTTCTTCCAGTGTGTGGGTTCAGGAACAGAGTCGCCTGCCTCAACTGCTGCACTGAGGACCACTCCCTCCCCACCATCCCCAGGACCAGAGACCCCTCCAGCGGGAGTGCCACCTGCTGCTTCCTCCCAGGTGTGGGCTGCAGGACCAGCTGCCCAGGAATGGCTTTCTCGGGACCTCCTGCACCGGCCTTCCGACGCACTGGCCAAAAAGGGGCTTGGACCATTCCTGCAAACAGCCAAACCGGCGAGAAGAGGCCAGACATCTGCCTCCATTCTCCCCAGAGTGGTGCAAGCTCAGCGAGGTCCCCAGCCTCCCCCAGGGGAAGCAGGGATCCCTGGACACCCCACACCTCCAGCCACGCTCCCCTCGGAGCCTGTAGAGGGTGTCCAGGCTAGTCCCTGGCGGCCACGTCCAGTCTTGCCAACGCACCCGGCTCTGACCCTGCCCGTGTCCTCAGATGCCTCCTCTCCTTCACCGCCAGCCCCGAGGCCTGAACGACCTGAATCACTTCTGGTCTCAGGACCATCTGTCACCCTGACTGAAGGTCTAGGAACTGTGAGGCCTGAACAGGACCCCGCCAAGTCTCCAGGAAGTCCCCTCCTGCTGAGAGGCTTGTCAAGCGGGGATGTGGCTGCACCTGAGCCCATCATGGGGGAGCCCGGCCAAGCCAGTGAGGAGTTCCAGCCATTGGCGAGGCCCTGGCGGGCCACACTGGCTGCAGAGGAGCTGGTTTCTCACCGTTCTCCCGGAGAGCCCCAGGAAACGTGCTCTGGAACGGAGGTGGAGAGGCCACGCCAGACAGGGCCTGGTCTCCCCAGGGAGGGGGCCAGGGGGCACATGGACCTTTCATCCTCAGAACCAAGCCAGGACATAGAGGGGCCGGGACTCTCCATCCTGCCAGCGAGGGATGCCACATTCTCCACCCCAAGTGTGAGGCAGCCAGACCCCAGTGCCTGGCTGAGTTCAGGACCTGAACTCACCGGGATGCCCAGGGTGAGGCTGGCAGCGCCCCTGGCAGTTCTTCCTATGGAACCTCTGCCACCAGAACCTGTTCGCCCAGCAGCTCTTCTGACACCCGAAGCCTCATCTGTGGGAGGGCCAGACCAGGCCCGATACCTGGAGTCAGCCCCTGGCTGGCCTGTGGGCCAGGAGGAGTGGGGGGTTGCACACACGTCCAGCCCTCCATCCACGCAAACCCTGAGCCTGTGGGCTCCCACAGGAGTGTTGCTACCCAGCCTGGTGGAGCTTGAATACCCCTTCCAGGCTGGCCGGGGGGCCTCACTCCAGCAGGAGCTGACAGAGCCCACCTTGGCCCTCAGTGCTGAAAGCCACAGGCCTCCTGAGCTTCAAGACAGTGTGGAGGGGCTTTCTGAGAGGCCCTCAC GC.

In some embodiments, a C1ORF127 has a nucleotide sequence having atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO: 1.

In some embodiments, a C1ORF127 gene product has an amino acid sequenceof SEQ ID NO: 2:

MGLERSDRYIMKCPMLRSRLGQESVHCGPMFIQVSRPLPLWRDNRQTPWLLSLRGELVASLEDASLMGLYVDMNATTVTVQSPRQGLLQRWEVLNTSAELLPLWLVSGHHAYSLEAACPPVSFQPESEVLVHIPKQRLGLVKRGSYIEETLSLRFLRVHQSNIFMVTENKDFVVVSIPAAGVLQVQRCQEVGGTPGTQAFYRVDLSLEFAEMAAPVLWTVESFFQCVGSGTESPASTAALRTTPSPPSPGPETPPAGVPPAASSQVWAAGPAAQEWLSRDLLHRPSDALAKKGLGPFLQTAKPARRGQTSASILPRVVQAQRGPQPPPGEAGIPGHPTPPATLPSEPVEGVQASPWRPRPVLPTHPALTLPVSSDASSPSPPAPRPERPESLLVSGPSVTLTEGLGTVRPEQDPAKSPGSPLLLRGLSSGDVAAPEPIMGEPGQASEEFQPLARPWRATLAAEELVSHRSPGEPQETCSGTEVERPRQTGPGLPREGARGHMDLSSSEPSQDIEGPGLSILPARDATFSTPSVRQPDPSAWLSSGPELTGMPRVRLAAPLAVLPMEPLPPEPVRPAALLTPEASSVGGPDQARYLESAPGWPVGQEEWGVAHTSSPPSTQTLSLWAPTGVLLPSLVELEYPFQAGRGASLQQELTEPTLALSAESHRPPELQDSVEGLSERPSR.

In some embodiments, C1ORF127 gene product has an amino acid sequencehaving at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 2.

In some embodiments, the agent comprises a conserved domain of theC1ORF127 gene product or a functional portion or functional variantthereof. In some embodiments, the conserved domain has the amino acidsequence of SEQ ID NO: 3:

KCPMLRSRLGQESVHCGPMFIQVSRPLPLWRDNRQTPWLLSLRGELVASLEDASLMGLYVDMNATTVTVQSPRQGLLQRWEVLNTSAELLPLWLVSGHHAYSLEAACPPVSFQPESEVLVHIPKQRLGLVKRGSYIEETLSLRFLRVHQSNIFMVTENKDEVVVSIPAAGVLQVQRCQEVGGTPGTQAFYRVDLSLEFAE MAAPVLWTVESFFQC

In some embodiments, the agent comprises a portion of the conserveddomain corresponding to SEQ ID NO: 4 or a functional portion orfunctional variant thereof:

GSYIEETLSLRFLRVHQSNIFMVTENKDFVVVSIPAAGVLQVQRCQEVGGTPGTQAFYRVDLSLEFAEMAAPVLWTVESFFQC

In some embodiments, the agent comprises an approximately 57 kDa proteinproduced by cleavage of the C1ORF127 gene product or a functionalportion or functional variant thereof. In some embodiments, theapproximately 57 kDa protein has the amino acid sequence of SEQ ID NO:5:

GSYIEETLSLRFLRVHQSNIFMVTENKDFVVVSIPAAGVLQVQRCQEVGGTPGTQAFYRVDLSLEFAEMAAPVLWTVESFFQCVGSGTESPASTAALRTTPSPPSPGPETPPAGVPPAASSQVWAAGPAAQEWLSRDLLHRPSDALAKKGLGPFLQTAKPARRGQTSASILPRVVQAQRGPQPPPGEAGIPGHPTPPATLPSEPVEGVQASPWRPRPVLPTHPALTLPVSSDASSPSPPAPRPERPESLLVSGPSVTLTEGLGTVRPEQDPAKSPGSPLLLRGLSSGDVAAPEPIMGEPGQASEEFQPLARPWRATLAAEELVSHRSPGEPQETCSGTEVERPRQTGPGLPREGARGHMDLSSSEPSQDIEGPGLSILPARDATFSTPSVRQPDPSAWLSSGPELTGMPRVRLAAPLAVLPMEPLPPEPVRPAALLTPEASSVGGPDQARYLESAPGWPVGQEEWGVAHTSSPPSTQTLSLWAPTGVLLPSLVELEYPFQAGRGASLQQELTEPTLALSAESHRPPELQDSVEGLSERPSR

In some embodiments, the C1ORF127 gene product has an amino acidsequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to SEQ ID NO: 3, SEQ ID NO: 4, or SEQ IDNO: 5.

In some embodiments, the agent comprises a nucleotide sequence thatcodes for the C1ORF127 gene product of SEQ ID NO: 3, SEQ ID NO: 4, orSEQ ID NO: 5, or a functional portion or functional variant thereof. Insome embodiments, the agent comprises a nucleotide sequence that codesfor a polypeptide that has at least about 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3, SEQ ID NO: 4,or SEQ ID NO: 5, or a functional portion or functional variant thereof.

In some embodiments, the agent comprises C1ORF127 or a C1ORF127 geneproduct (e.g., C1ORF127 protein). In some embodiments, the agent is aC1ORF127 gene product having at least one different post-translationalmodification than a native C1ORF127 gene product. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation (e.g., O-linked oligosaccharides, N-linkedoligosaccharides, etc.), phosphorylation, lipidation, and acylation. Insome embodiments, the agent comprises a C1ORF127 gene product having atleast one substituted, deleted, or added amino acid than a nativeC1ORF127 gene product. In some embodiments, the C1ORF127 gene producthas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more substituted,deleted, or added amino acids than a native C1ORF127 gene product. Insome embodiments, the C1ORF127 gene product has 1, 2, 3, 4, or 5 lesscysteines than native C1ORF127 gene product. In some embodiments, the 1,2, 3, 4, or 5 less cysteines correspond to the conserved cysteines asshown in SEQ ID NO: 3.

In some embodiments, the C1ORF127 gene product is differentlyphosphorylated than naturally occurring C1ORF127 gene product. In someembodiments, the C1ORF127 gene product has one less phosphorylation thannaturally occurring C1ORF127 gene product. In some embodiments, theC1ORF127 gene product has one more phosphorylation than naturallyoccurring C1ORF127 gene product. In some embodiments, the differentphosphorylation is present in the portion of C1ORF127 gene productcorresponding to SEQ ID NO: 4 or SEQ ID NO: 5 (e.g., in a putativephosphorylation site in SEQ ID NO: 4 or SEQ ID NO: 5).

In some embodiments, the C1ORF127 gene product is differentlyglycosylated than naturally occurring C1ORF127 gene product. In someembodiments, the C1ORF127 gene product has one less glycosylation thannaturally occurring C1ORF127 gene product. In some embodiments, theC1ORF127 gene product has two less glycosylations than naturallyoccurring C1ORF127 gene product. In some embodiments, the C1ORF127 geneproduct has one more glycosylation than naturally occurring C1ORF127gene product. In some embodiments, the C1ORF127 gene product has twomore glycosylations than naturally occurring C1ORF127 gene product. Insome embodiments, the different glycosylation is present in the portionof C1ORF127 gene product corresponding to SEQ ID NO: 4 or SEQ ID NO: 5(e.g., in a putative glycosylation site in SEQ ID NO: 4 or SEQ ID NO:5).

In some embodiments, the C1ORF127 gene product is a dimer, trimer, ormultimer. In some embodiments, the C1ORF127 gene product is a homodimer,homotrimer, or homomultimer. In some embodiments, the C1ORF127 geneproduct exists in a different multimerization state than naturallyoccurring C1ORF127 gene product.

In some embodiments, the agent comprises a C1ORF127 gene product havinga different composition, activity or activity level than native C1ORF127gene product. In some embodiments, the C1ORF127 gene product has a bloodglucose clearance activity that is about 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2.0-fold, 2.5-fold,3.0-fold, or more than a native C1ORF127 gene product. In someembodiments, the C1ORF127 gene product has a blood glucose clearanceactivity that is about 1%, 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50% orless than a native C1ORF127 gene product. In some embodiments, the agentcomprises a functional portion (i.e., functional fragment) of a C1ORF127gene product. In some embodiments, the agent comprises a functionalfragment of C1ORF127 gene product corresponding to SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5, or a polypeptide sequence having at least about80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

In some embodiments, the agent comprises a C1ORF127 gene productcomprising a furin cleavage site. In some embodiments, the agentcomprises a C1ORF127 gene product without a PC2 cleavage site (e.g.,LVKRG in SEQ ID NO: 2). In some embodiments, the agent comprises aC1ORF127 gene product with a furin cleavage site and without a PC2cleavage site.

In some embodiments, the agent is a cell expressing C1ORF127 geneproduct. In some embodiments, the cell is an islet cell or a beta-cell.In some embodiments, the cell is a pancreatic delta cells. In someembodiments, the cell is autologous to the subject requiring treatment.In some embodiments, the cell is stem cell derived. In some embodiments,the stem cell derived cell is a stem cell derived beta cell. Methods ofderiving beta cells are taught in the art. See, e.g., WO 2015/002724published Jan. 8, 2015, herein incorporated by reference in itsentirety. In some embodiments, the cell is encased in a microcapsule orsemi-permeable membrane.

Aspects of the disclosure involve microcapsules comprising isolatedpopulations of cells described herein, e.g., cells expressing a C1ORF127gene product or functional variant or functional fragment thereof.Microcapsules are well known in the art. Suitable examples ofmicrocapsules are described in the literature (e.g., Jahansouz et al.,“Evolution of β-Cell Replacement Therapy in Diabetes Mellitus: IsletCell Transplantation” Journal of Transplantation 2011; Volume 2011,Article ID 247959; Orive et al., “Application of cell encapsulation forcontrolled delivery of biological therapeutics”, Advanced Drug DeliveryReviews (2013), http://dx.doi.org/10.1016/j.addr.2013.07.009; Hernandezet al., “Microcapsules and microcarriers for in situ cell delivery”,Advanced Drug Delivery Reviews 2010; 62:711-730; Murua et al., “Cellmicroencapsulation technology: Towards clinical application”, Journal ofControlled Release 2008; 132:76-83; and Zanin et al., “The developmentof encapsulated cell technologies as therapies for neurological andsensory diseases”, Journal of Controlled Release 2012; 160:3-13).Microcapsules can be formulated in a variety of ways. Exemplarymicrocapsules comprise an alginate core surrounded by a polycation layercovered by an outer alignate membrane. The polycation membrane forms asemipermeable membrane, which imparts stability and biocompatibility.Examples of polycations include, without limitation, poly-L-lysine,poly-L-ornithine, chitosan, lactose modified chitosan, andphotopolymerized biomaterials. In some embodiments, the alginate core ismodified, for example, to produce a scaffold comprising an alginate corehaving covalently conjugated oligopeptides with an RGD sequence(arginine, glycine, aspartic acid). In some embodiments, the alginatecore is modified, for example, to produce a covalently reinforcedmicrocapsule having a chemoenzymatically engineered alginate of enhancedstability. In some embodiments, the alginate core is modified, forexample, to produce membrane-mimetic films assembled by in-situpolymerization of acrylate functionalized phospholipids. In someembodiments, microcapsules are composed of enzymatically modifiedalginates using epimerases. In some embodiments, microcapsules comprisecovalent links between adjacent layers of the microcapsule membrane. Insome embodiment, the microcapsule comprises a subsieve-size capsulecomprising aliginate coupled with phenol moieties. In some embodiments,the microcapsule comprises a scaffold comprising alginate-agarose. Insome embodiments, the cell is modified with PEG before beingencapsulated within alginate. In some embodiments, the isolatedpopulations of cells are encapsulated in photoreactive liposomes andalginate. It should be appreciate that the alginate employed in themicrocapsules can be replaced with other suitable biomaterials,including, without limitation, PEG, chitosan, PES hollow fibers,collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBV andPVA, PGSAS, agarose, agarose with gelatin, PLGA, and multilayerembodiments of these.

In some embodiments, the agent further comprises a pharmaceuticallyacceptable carrier or excipient. As used herein, “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Suitable carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.Preferred examples of such carriers or diluents include, but are notlimited to, water, saline, finger's solutions, dextrose solution, and 5%human serum albumin. Liposomes and non-aqueous vehicles such as fixedoils may also be used. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the agents/compositions is contemplated.Supplementary active compounds can also be incorporated into theagents/compositions.

In some embodiments, the methods disclosed herein further comprisesadministration to the subject of one or more additional anti-diabetictherapeutics (e.g., Acarbose (Precose), Albiglutide (Tanzeum),Alogliptin (Nesina), Alogliptin and metformin (Kazano), Alogliptin andpioglitazone (Oseni), Bromocriptine mesylate (Cycloset, Parlodel),Canaglifozin (Invokana), Canagliflozin and metformin (Invokamet),Dapagliflozin (Farxiga), Dapagliflozin and metformin (Xigduo XR),Dulaglutide (Trulicity), Empagliflozin (Jardiance), Empagliflozin andlinagliptin (Glyxambi), Empagliflozin and metformin (Synjardy),Exenatide Byetta), Glimepiride (Amaryl), Glyburide (DiaBeta, Glynase),Glyburide and metformin (Glucovance) Insulin aspart (NovoLog), Insulindegludec (Tresiba), Insulin glargine (Basaglar, Lantus, Toujeo), InsulinIsophane (Humulin N, Novolin N), Insulin Isophane/regular insulin(Humulin 70/30, Novolin 70/30), Insulin lispro (Humalog), Linagliptin(Tradjenta), Liraglutide (Victoza), Metformin (Glucophage), Miglitol(Glyset), Nateglinide (Starlix), Pioglitazone (Actos), Repaglinide(Prandin), Rosiglitazone Avandia), Rosiglitazone and glimepiride(Avandaryl), Rosiglitazone and metformin (Avandamet), Saxagliptin(Onglyza), Semaglutide (Ozempic), or Sitagliptin (Januvia)).

In some embodiments, administration of the agent improves blood glucoseclearance. In some embodiments, administration of the agent returnsblood glucose levels from a pathological level to a non-pathologicallevel (e.g., from a hyperglycemic state to a normal state). In someembodiments, administration of the agent reduces blood glucose levels byabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or more. In someembodiments, administration of the agent reduces blood glucose levels toabout less than 140 mg/dL, about less than 100 mg/dL, about 60-90 mg/dL,or about 70-80 mg/dL. In some embodiments, administration of the agentdoes not cause hypoglycemia in the subject (e.g., a blood sugar of lessthan about 70 mg/dL, a blood sugar of less than about 60 mg/dL, or to ablood sugar level wherein the subject does not exhibit signs ofhypoglycemia). In some embodiments, the blood glucose clearance propertyof the agent is independent of insulin activity.

In some embodiments, the agent has glucose sensitizer activity whenadministered to the subject. In some embodiments, the agent increasesinsulin activity, production or secretion when administered to asubject. In some embodiments, administration of the agent increasesinsulin activity, production or secretion by about 5%, 10%, 20%, 30%,40%, 50%, 75%, 100%, or more. In some embodiments, administration of theagent increases insulin activity, production or secretion by about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 5-fold, 10-fold, 50-fold, or more.

In some embodiments, the agent increases the rate of glucose turnoverwhen administered to the subject. In some embodiments, administration ofthe agent increases glucose turnover by about 5%, 10%, 20%, 30%, 40%,50%, 75%, 100%, or more. In some embodiments, administration of theagent increases glucose turnover about 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,5-fold, 10-fold, 50-fold, or more.

In some embodiments, the agent increases glycolysis when administered tothe subject. In some embodiments, the agent increases the rate ofglycolysis when administered to the subject. In some embodiments,administration of the agent increases glycolysis by about 5%, 10%, 20%,30%, 40%, 50%, 75%, 100%, or more. In some embodiments, administrationof the agent increases glycolysis about 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,5-fold, 10-fold, 50-fold, or more.

In some embodiments, the agent increases the rate of glycogen synthesiswhen administered to the subject. In some embodiments, the agentincreases the rate of glycogen synthesis when administered to thesubject. In some embodiments, administration of the agent increasesglycogen synthesis by about 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, ormore. In some embodiments, administration of the agent increasesglycogen synthesis about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 5-fold, 10-fold,50-fold, or more. In some embodiments, the agent has glucagon-likeactivity when administered to the subject.

In some embodiments, the subject has diabetes (e.g., Type I diabetes orType II diabetes), metabolic syndrome, glucose intolerance, or obesity.In some embodiments, the subject has diabetes. In some embodiments, thesubject's genome comprises a mutant or variant form of C1ORF127. In someembodiments, the variant is one of the following: 1_11014118_C_T,1_11015165_A_G, 1_11008102_G_A, 1_11009679_G_A, 1_11007881_G_T,1_11008778_T_A,C, 1_11008799_G_C, 1_11008417_G_A, 1_11008127_C_T,1_11009703_C_T, 1_11008594_A_T, 1_11007997_C_T, 1_11024271_G_T,A,1_11008685_T_C, 1_11009716_C_G, 1_11009844 G_A, 1_11008417_G_A,1_11036248_G_A, 1_11024271_G_T,A, 1_11009844_G_A, 1_11008799_G_C,1_11008778_T_A,C, 1_11008685_T_C, 1_11014118_C_T, 1_11009716_C_G,1_11008102_G_A, 1_11007895_G_T, 1_11008127_C_T, 1_11009703_C_T,1_11008594_A_T, 1_11009679_G_A, 1_11014127_C_T, 1_11008844_C_G,T,1_11015165_A_G, 1_11009703_C_T, 1_11009679_G_A, 1_11008594_A_T,1_11008102_GA, 1_11009844_G_A, 1_11007895_G_T, 1_11008127_C_T,1_11008417_G_A, 1_11007724_C_T, 1_11024271_G_T,A, 1_11008778_T_A,C,1_11009716_C_G, 1_11015165_A_G, 1_11014118_C_T, 1_11008799_G_C,1_11007881_G_T, 1_11008685_T_C, 1_11007997_C_T, 1_11015165_A_G,1_11008102_G_A, 1_11008685_T_C, 1_11008799_G_C, 1_11008127_C_T,1_11009679_G_A, 1_11014118_C_T, 1_11009703_C_T, 1_11007724_C_T,1_11008594_A_T, 1_11009716_C_G, 1_11007895 G_T, 1_11007881_G_T,1_11008778_T_A,C, 1_11024271_G_T,A, or 11007997_C_T.

In some embodiments, the C1ORF127 variant is associated with diabetesand high BMI. In some embodiments, the C1ORF127 variant associated withdiabetes and high BMI is 1_11033415_G_A. In some embodiments, theC1ORF127 variant is associated with elevated fasting glucose levels. Insome embodiments, the C1ORF127 variant associated with elevated fastingglucose levels is 1_11014118_C_T. In some embodiments, the variant isassociated with type 1 diabetes. In some embodiments, the variant isassociated with type 2 diabetes. In some embodiments, the variant isassociated with high BMI.

In some embodiments, administration of the agent corrects a geneticdefect in the subject causing aberrant expression or activity of theC1ORF127 gene product. In some embodiments, the genetic defect is aC1ORF127 variant as identified herein. In some embodiments, the geneticdefect is variant 1_11033415_G_A. In some embodiments, the geneticdefect is variant 1_11014118_C_T. In some embodiments, the agentcomprises a targetable nuclease. In some embodiments, the agent furthercomprises gRNA targeting the nuclease to the genetic defect. In someembodiments, the agent further comprises a donor nucleic acid forcorrecting the defect by homology directed repair (HDR) after thegeneration of a DNA break at the defect site by the targetable nuclease.

Targetable nucleases (e.g., site specific nucleases) generate DNA breaksin the genome at a selected target site and can be used to produceprecise genomic modifications. DNA breaks, e.g., double-stranded DNAbreaks, can be repaired by various DNA repair pathways. Homologousrecombination (HR) mediated repair (also termed homology-directed repair(HDR)) uses homologous donor DNA as a template to repair the break. Ifthe sequence of the donor DNA differs from the genomic sequence, thisprocess leads to the introduction of sequence changes into the genome.Precise modifications to the genome can be made by providing donor DNAcomprising an appropriate sequence. Modifications that can be generatedusing targetable nucleases include insertions, deletions, orsubstitutions of one or more nucleotides, or introducing an exogenousDNA segment such as an expression cassette (a nucleic acid comprising asequence to be expressed and appropriate expression control elements,such as a promoter, to cause the sequence to be expressed in a cell) ortag at a selected location in the genome.

There are currently four main types of targetable nuclease in use: zincfinger nucleases (ZFNs), transcription activator-like effector nucleases(TALENs), and RNA-guided nucleases (RGNs) such as the Cas proteins ofthe CRISPR/Cas Type II system, and engineered meganucleases. ZFNs andTALENs comprise the nuclease domain of the restriction enzyme FokI (oran engineered variant thereof) fused to a site-specific DNA bindingdomain (DBD) that is appropriately designed to target the protein to aselected DNA sequence. In the case of ZFNs, the DNA binding domaincomprises a zinc finger DBD. In the case of TALENs, the site-specificDBD is designed based on the DNA recognition code employed bytranscription activator-like effectors (TALEs), a family ofsite-specific DNA binding proteins found in plant-pathogenic bacteriasuch as Xanthomonas species. The Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) Type II system is a bacterial adaptiveimmune system that has been modified for use as an RNA-guidedendonuclease technology for genome engineering. The bacterial systemcomprises two endogenous bacterial RNAs called crRNA and tracrRNA and aCRISPR-associated (Cas) nuclease, e.g., Cas9. The tracrRNA has partialcomplementarity to the crRNA and forms a complex with it. The Casprotein is guided to the target sequence by the crRNA/tracrRNA complex,which forms a RNA/DNA hybrid between the crRNA sequence and thehomologous sequence in the target. For use in genome modification, thecrRNA and tracrRNA components are often combined into a single chimericguide RNA (sgRNA or gRNA) in which the targeting specificity of thecrRNA and the properties of the tracrRNA are combined into a singletranscript that localizes the Cas protein to the target sequence so thatthe Cas protein can cleave the DNA. The sgRNA often comprises anapproximately 20 nucleotide guide sequence complementary to the desiredtarget sequence followed by about 80 nt of hybrid crRNA/tracrRNA. One ofordinary skill in the art appreciates that the guide RNA need not beperfectly complementary to the target sequence. For example, in someembodiments it may have one or two mismatches.

In some embodiments, one or more guide sequences (e.g., guide RNA, gRNA)is a naturally occurring RNA sequence, a modified RNA sequence (e.g., aRNA sequence comprising one or more modified bases), a synthetic RNAsequence, or a combination thereof. As used herein a “modified RNA” isan RNA comprising one or more modifications (e.g., RNA comprising one ormore non-standard and/or non-naturally occurring bases and/ormodifications to the backbone, internucleoside linkage(s) and/or sugar).Methods of modifying bases of RNA are well known in the art. Examples ofsuch modified bases include those contained in the nucleosides5-methylcytidine (5mC), pseudouridine (Ψ), 5-methyluridine,2′0-methyluridine, 2-thiouridine, N-6 methyladenosine, hypoxanthine,dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). It shouldbe noted that any number of bases, sugars, or backbone linkages in a RNAsequence can be modified in various embodiments. It should further beunderstood that combinations of different modifications may be used. Insome embodiments an RNA comprises one or more modifications selectedfrom: phosphorothioate, 2′-OMe, 2′-F, 2′-constrained ethyl (2′-cEt),2′-OMe 3′ phosphorothioate (MS), and 2′-OMe 3-thioPACE (MSP)modifications. In some embodiments a modification may stabilize the RNAand/or increase its binding affinity to a complementary sequence.

In some embodiments, the one or more guide sequences comprise at leastone locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8LNA units, such as from about 3-7 or 4-8 LNA units, or 3, 4, 5, 6 or 7LNA units. In some embodiments, all the nucleotides of the one or moreguide sequences are LNA. In some embodiments, the one or more guidesequences may comprise both beta-D-oxy-LNA, and one or more of thefollowing LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in eitherthe beta-D or alpha-L configurations or combinations thereof. In someembodiments all LNA cytosine units are 5′methyl-cytosine.

In some embodiments, the one or more guide sequences is a morpholino.Morpholinos are typically synthetic molecules, of about 25 bases inlength and bind to complementary sequences of RNA by standard nucleicacid base-pairing. Morpholinos have standard nucleic acid bases, butthose bases are bound to morpholine rings instead of deoxyribose ringsand are linked through phosphorodiamidate groups instead of phosphates.

In some embodiments, a guide sequence can vary in length from about 8base pairs (bp) to about 200 bp. In some embodiments, each of one ormore guide sequences can be about 9 to about 190 bp; about 10 to about150 bp; about 15 to about 120 bp; about 20 to about 100 bp; about 30 toabout 90 bp; about 40 to about 80 bp; about 50 to about 70 bp in length.

Chemical modifications and methods of synthesizing guide RNAs (guidesequences) are known in the art. See WO/2016/164356, herein incorporatedby reference in its entirety.

The portion of each genomic sequence (e.g., target sequence of interest,gene of interest, genetic defect) to which each guide sequence iscomplementary or homologous to can also vary in size. In particularaspects, the portion of each genomic sequence to which the guidesequence is complementary or homologous to can be about 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38 39, 40, 41, 42, 43, 44, 45, 46 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 81, 82, 83,84, 85, 86, 87 88, 89, 90, 81, 92, 93, 94, 95, 96, 97, 98, or 100nucleotides (contiguous nucleotides) in length. In some embodiments,each guide sequence can be at least about 70%, 75%, 80%, 85%, 90%, 95%,100%, etc. identical, complementary or similar to the portion of eachgenomic sequence. In some embodiments, each guide sequence is completelyor partially identical, complementary or similar to each genomicsequence. For example, each guide sequence can differ from perfectcomplementarity or homology to the portion of the genomic sequence byabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, etc. nucleotides. In some embodiments, one or more guide sequencesare perfectly complementary or homologous (100%) across at least about10 to about 25 (e.g., about 20) nucleotides of the genomic sequence.

The genomic target sequence (e.g., genomic locus of interest, gene ofinterest, target sequence of interest, genetic defect) should also beimmediately followed by a Protospacer Adjacent Motif (PAM) sequence. ThePAM sequence is present in the DNA target sequence but not in a guidesequence. The Cas protein will be directed to any DNA sequence with thecorrect target sequence followed by the PAM sequence. The PAM sequencevaries depending on the species of bacteria from which the Cas proteinwas derived. In some embodiments, the targetable nuclease comprises aCas9 protein. For example, Cas9 from Streptococcus pyogenes (Sp),Neisseria meningitides, Staphylococcus aureus, Streptococcusthermophiles, or Treponema denticola may be used. The PAM sequences forthese Cas9 proteins are NGG, NNNNGATT, NNAGAA, NAAAAC, respectively. Anumber of engineered variants of the site-specific nucleases have beendeveloped and may be used in certain embodiments. For example,engineered variants of Cas9 and Fok1 are known in the art. Furthermore,it will be understood that a biologically active fragment or variant canbe used. Other variations include the use of hybrid targetablenucleases. For example, in CRISPR RNA-guided FokI nucleases (RFNs) theFokI nuclease domain is fused to the amino-terminal end of acatalytically inactive Cas9 protein (dCas9) protein. RFNs act as dimersand utilize two guide RNAs (Tsai, Q S, et al., Nat Biotechnol. 2014;32(6): 569-576). Site-specific nucleases that produce a single-strandedDNA break are also of use for genome editing. Such nucleases, sometimestermed “nickases” can be generated by introducing a mutation (e.g., analanine substitution) at key catalytic residues in one of the twonuclease domains of a targetable nuclease that comprises two nucleasedomains (such as ZFNs, TALENs, and Cas proteins). Examples of suchmutations include D10A, N863A, and H840A in SpCas9 or at homologouspositions in other Cas9 proteins. A nick can stimulate HDR at lowefficiency in some cell types. Two nickases, targeted to a pair ofsequences that are near each other and on opposite strands can create asingle-stranded break on each strand (“double nicking”), effectivelygenerating a DSB, which can be repaired by HDR using a donor DNAtemplate (Ran, F. A. et al. Cell 154, 1380-1389 (2013).

The term “donor nucleic acid” or “donor” refers to an exogenous nucleicacid segment that, when provided to a cell, e.g., along with atargetable nuclease, can be used as a template for DNA repair byhomologous recombination and thereby cause site-specific genomemodification (sometimes termed “genome editing”). The modifications caninclude insertions, deletions, or substitutions of one or morenucleotides, or introducing an exogenous DNA segment such as anexpression cassette or tag at a selected location in the genome. A donornucleic acid typically comprises sequences that have homology to theregion of the genome at which the genomic modification is to be made.The donor may contain one or more single base changes, insertions,deletions, or other alterations with respect to the genomic sequence, solong as it has sufficient homology to allow for homology-directedrepair. In the present invention, the donor nucleic acid is the nucleicacid sequence comprising the reporter gene and WPRE flanked by thehomology arms. The homology arms are homologous to genomic sequencesflanking a location in genomic DNA at which the insertion is to be made(e.g., DNA break). One of ordinary skill in the art also appreciatesthat the homology need not extend all the way to the DNA break. Forexample, in some embodiments the homology begins no more than 100 bpaway from the break, e.g., between 1 and 100 bp away, e.g., 1-50 bpaway, e.g., 1-15 bp away, from the break.

Donor nucleic acid can be provided, for example, in the form of DNAplasmids, PCR products, or chemically synthesized oligonucleotides, andmay be double-stranded or single-stranded in various embodiments. Thesize of the donor nucleic can vary from as small as about 40 base pairs(bp) to about 10 kilobases (kb), or more. In some embodiments the donornucleic is between about 1 kb and about 5 kb long.

Those of ordinary skill in the art are aware of methods for performingsite-specific genome modification using targetable nucleases and will beable to apply such methods to repair a genetic defect associated withaberrant expression or activity of C1ORF127 as taught herein. Those ofordinary skill in the art can, for example, design appropriate guideRNAs, TALENs, or ZFNs to generate a DNA break at a selected location inthe genome, can design a targeting vector (e.g., comprising homologyarms) to promote HDR at a DNA break generated by a targetable nuclease,and are aware of appropriate methods that can be used to introduce atargetable nuclease into cells and, where appropriate, a donor nucleicacid, and/or guide RNA. A targetable nuclease may be targeted to aunique site in the genome of a mammalian cell by appropriate design ofthe nuclease or guide RNA. A nuclease or guide RNA may be introducedinto cells by introducing a nucleic acid that encodes it into the cell.Standard methods such as plasmid DNA transfection, viral vectordelivery, transfection with synthetic mRNA (e.g., capped, polyadenylatedmRNA), or microinjection can be used. If DNA encoding the nuclease orguide RNA is introduced, the coding sequences should be operably linkedto appropriate regulatory elements for expression, such as a promoterand termination signal. In some embodiments a sequence encoding a guideRNA is operably linked to an RNA polymerase III promoter such as U6 ortRNA promoter. In some embodiments one or more guide RNAs and Casprotein coding sequences are transcribed from the same nucleic acid(e.g., plasmid). In some embodiments multiple guide RNAs are transcribedfrom the same plasmid or from different plasmids or are otherwiseintroduced into the cell. The multiple guide RNAs may direct Cas9 todifferent target sequences in the genome, allowing for multiplexedgenome editing. In some embodiments a nuclease protein (e.g., Cas9) maycomprise or be modified to comprise a nuclear localization signal (e.g.,SV40 NLS). A nuclease protein may be introduced into cells, e.g., usingprotein transduction. Nuclease proteins, guide RNAs, or both, may beintroduced using microinjection. Methods of using targetable nucleases,e.g., to perform genome editing, are described in numerous publications,such as Methods in Enzymology, Doudna J A, Sontheimer E J. (eds), Theuse of CRISPR/Cas9, ZFNs, and TALENs in generating site-specific genomealterations. Methods Enzymol. 2014, Vol. 546 (Elsevier); Carroll, D.,Genome Editing with Targetable Nucleases, Annu. Rev. Biochem. 2014.83:409-39, and references in either of these. See also U.S. Pat. Pub.Nos. 20140068797, 20140186919, 20140170753 and/or PCT/US2014/034387(WO/2014/172470). Each of these references is incorporated by referencein its entirety.

Agents and Compositions

Some aspects of the disclosure are related to agents that increase thelevel or activity of a C1ORF127 gene product when administered to asubject. The agents may be any agent as described herein. The C1ORF127gene product may be any C1ORF127 gene product as described herein. Insome embodiments, the agent increases the level or activity ofendogenous C1ORF127 gene product when administered to a subject. In someembodiments, the agent modulates (e.g., increases or decreases) theexpression of endogenous C1ORF127 gene product when administered to asubject. The agent may increase or decrease expression by any amount andis not limited. In some embodiments, the agent increases expression byabout 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 2.0-fold, 2.5-fold, 3.0-fold, 5-fold, or more. Insome embodiments, the agent increases expression or decreases expressionby about 1%, 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 200%, 300%, 500%, or more.

In some embodiments, the agent modulates (e.g., increases or decreases)the secretion of endogenous C1ORF127 gene product when administered to asubject. The agent may increase or decrease secretion by any amount andis not limited. In some embodiments, the agent increases secretion byabout 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 2.0-fold, 2.5-fold, 3.0-fold, 5-fold, or more. Insome embodiments, the agent increases secretion or decreases secretionby about 1%, 2.5%, 5%, 7.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 200%, 300%, 500%, or more.

In some embodiments, the agent comprises a small molecule, a protein, ora nucleic acid. In some embodiments, the agent comprises C1ORF127 geneproduct having at least one different post-translational modificationthan native C1ORF127 gene product. In some embodiments, the agentcomprises C1ORF127 gene product having at least one substituted,deleted, or added amino acid than native C1ORF127 gene product. In someembodiments, the agent comprises C1ORF127 gene product having adifferent activity or activity level than native C1ORF127 gene product.In some embodiments, wherein the agent comprises a functional portion ofthe C1ORF127 gene product.

In some embodiments, the agent further comprises a pharmaceuticallyacceptable carrier or excipient. The pharmaceutically acceptable carrieror excipient is not limited and may be any pharmaceutically acceptablecarrier or excipient described herein.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose, for example. The term “unitdose” when used in reference to a therapeutic composition refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required physiologically acceptable diluent, i.e., carrier, orvehicle.

The dosage ranges for the agent depends upon the potency, and areamounts large enough to produce the desired effect e.g., improve bloodglucose clearance. The dosage should not be so large as to causeunacceptable adverse side effects. Generally, the dosage will vary withthe age, condition, and sex of the patient and can be determined by oneof skill in the art. The dosage can also be adjusted by the individualphysician in the event of any complication. Typically, the dosage canrange from 0.001 mg/kg body weight to 0.5 mg/kg body weight. In oneembodiment, the dose range is from 5 μg/kg body weight to 30 μg/kg bodyweight.

Administration of the doses recited above can be repeated. In someembodiments, the doses are given once a day, or multiple times a day,for example, but not limited to, three times a day. In some embodiments,the doses recited above are administered daily for weeks or months. Theduration of treatment depends upon the subject's clinical progress andresponsiveness to therapy.

Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are particular to eachindividual. However, suitable dosage ranges for systemic application aredisclosed herein and depend on the route of administration. Suitableregimes for administration are also variable, but are typified by aninitial administration followed by repeated doses at one or moreintervals by a subsequent administration. Alternatively, continuousintravenous infusion sufficient to maintain concentrations in the bloodin the ranges specified for in vivo therapies are contemplated. In someembodiments, the dosage range is sufficient to maintain concentrationsin the blood in the range found in the blood of a population of normal,healthy human subjects.

In some embodiments, the agent comprises an additional anti-diabetictherapeutic. The additional anti-diabetic therapeutic is not limited andmay be any anti-diabetic therapeutic described herein. The additionalanti-diabetic therapeutic may be administered together or separately. Insome embodiments, the additional anti-diabetic therapeutic is in asingle dosage form. In some embodiments, the additional anti-diabetictherapeutic is in a separate dosage form.

In some embodiments, the agent improves blood glucose clearance whenadministered to a subject. In some embodiments, the agent does not causehypoglycemia when administered to a subject.

In some embodiments, the subject has diabetes (e.g., Type I diabetes orType II diabetes), metabolic syndrome, glucose intolerance, or obesity.In some embodiments, the subject has diabetes. In some embodiments, thesubject is human or murine.

In some embodiments, the agent comprises a C1ORF127 gene product of SEQID NO: 2 or a functional portion or functional variant thereof. In someembodiments, the agent comprises a nucleic acid coding for a C1ORF127gene product, a functional portion, or functional variant thereof,wherein the nucleic acid comprises the sequence of SEQ ID NO: 1 or aportion thereof. In some embodiments, the agent comprises a nucleic acidcoding for a C1ORF127 gene product a functional portion or functionalvariant thereof, wherein the nucleic acid comprises a sequence having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%homology to SEQ ID NO: 1 or a portion thereof.

In some embodiments, the agent corrects a genetic defect in the subjectcausing aberrant expression or activity of the C1ORF127 gene product. Insome embodiments, the agent comprises a targetable nuclease as describedherein. In some embodiments, the agent comprises guide RNA and,optionally, donor nucleic acid as described herein.

Methods of Diagnosing

Some aspects of the disclosure are directed to methods of diagnosing aC1ORF127-related disorder or an increased risk for developing aC1ORF127-related disorder in a test individual, comprising determining aC1ORF127 gene product level in a sample obtained from said testindividual, wherein a C1ORF127 gene product level that is increased ordecreased in said test individual compared to a C1ORF127 gene productlevel in a normal individual is indicative of a C1ORF127-relateddisorder. The test individual may be any subject described herein.

The level of C1ORF127 which is indicative of a C1ORF127-relatedcondition may be defined as the decreased level present in samples fromindividuals known to have a C1ORF127-related disorder over the C1ORF127level in samples from individuals known to be free of a C1ORF127-relateddisorder. The level of C1ORF127 may be, for example, at least 1.1 fold,1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold,1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold,2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold,3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold,4.0 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold,4.7 fold, 4.8 fold, 4.9 fold, 5.0 fold, 5.1 fold, 5.2 fold, 5.3 fold,5.4 fold, 5.5 fold, 5.6 fold, 5.7 fold, 5.8 fold, 5.9 fold, 6.0 fold, 10fold, 15 fold, 20 fold, 50 fold or 100 fold higher or lower in a samplefrom an individual with a C1ORF127-related disorder.

The C1ORF127 gene product (e.g., protein) is detected and/or quantifiedin the sample using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of general immunoassays, seealso Methods in Cell Biology Volume 37: Antibodies in Cell Biology,Asai, ed. Academic Press, Inc. New York (1993); Basic and ClinicalImmunology 7th Edition, Stites & Terr, eds. (1991).

In some embodiments, the C1ORF127 gene product in the sample can also bedetected and quantified using immunoblot (Western blot) analysis.Immunoblotting generally comprises separating sample proteins by gelelectrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support, (such as anitrocellulose filter, a nylon filter, or derivatized nylon filter), andincubating the sample with antibodies that specifically bind theC1ORF127 gene product. The anti-C1ORF127 gene product antibodiesspecifically bind to C1ORF127 gene product on the solid support. Theseantibodies may be directly labeled or alternatively may be subsequentlydetected using labeled antibodies (e.g., labeled sheep anti-mouseantibodies) that specifically bind to the anti-C1ORF127 gene productantibody.

In some embodiments, quantitative assays of C1ORF127 gene product aredeemed to show a positive result, e.g., elevated or decreased C1ORF127gene product level, when the measured C1ORF127 gene product level isgreater or less than the level measured or known for a control sample(e.g. either a level known or measured for a normal healthy individualor a “baseline/reference” level determined at a different time for thesame individual. In a particularly preferred embodiment, the assay isdeemed to show a positive result when the difference between sample and“control” is statistically significant (e.g. at the 85% or greater,preferably at the 90% or greater, more preferably at the 95% or greaterand most preferably at the 98% or greater confidence level).

In some embodiments, the C1ORF127 gene product level is detected in ablood sample. Methods of obtaining and processing the blood sample areknown in the art and are not limited herein.

Some aspects of the disclosure are directed to methods of diagnosing aC1ORF127-related disorder or an increased risk for developing aC1ORF127-related disorder in a test individual, comprising screening thetest individual for a mutation in C1ORF127. Methods of detecting geneticmutations are known in the art and not limited. In some embodiments, theC1ORF127-related disorder is diabetes.

Methods of Screening

Some aspects of the disclosure are directed to methods of screening fora C1ORF127 gene product receptor agonist, comprising contacting a cellresponsive to the C1ORF127 gene product with a test agent anddetermining the response of the cell, wherein if the cell responds thenthe test agent is identified as a C1ORF127 gene product receptoragonist. In some embodiments, the cell response is glucose uptake. Insome embodiments, the cell is further contacted with an insulin receptorantagonist. In some embodiments, the insulin receptor antagonist isS961. In some embodiments, an animal (e.g., a subject as describedherein) having the cell is used.

Methods for Enriching for mRNAs Coding for Secreted and Membrane BoundProteins

Some aspects of the disclosures are related to methods of enriching formRNAs coding for secreted and membrane bound proteins, comprising:providing a cell comprising a Endoplasmic Reticulum (ER) transloconhaving a label, performing sub-cellular fractionalization of the celland isolating an ER fraction containing the label, and isolating andsequencing mRNA contained in the isolated ER fraction containing thelabel.

The component of the ER translocon having a label is not limited. Insome embodiments, the labeled ER translocon component is Sec61, theoligosaccharyl transferase complex, the TRAP complex, or the membraneprotein TRAM. In some embodiments, the ER translocon component SEC61bhas the label.

Methods of adding a label to an ER translocon component are not limited.In some embodiments, the cell is genetically modified to express a labelwith the translocon component. The methods of genetic modification ofthe cell are not limited and any known in the art. In some embodiments,the cell is genetically using a targetable nuclease as described herein.In some embodiments, the label is a fluorescent label. In someembodiments the fluorescent label is a green fluorescent protein, redfluorescent protein, or infrared fluorescent protein.

In some embodiments, the label is transiently expressed or only undercertain cellular conditions. In some embodiments, the certain conditionsare the present of a site specific recombinase (e.g. Cre/Lox). Forinstance, the label can be added to the genome along with a stop codonflanked by LoxP. Upon activation/addition of Cre, the stop codon wouldbe removed during recombination and the label expressed along with thetranslocon component.

The term “site-specific recombinase” (also referred to simply as a“recombinase” herein) refers to a protein that can recognize andcatalyze the recombination of DNA between specific sequences in a DNAmolecule. Such sequences may be referred to as “recombination sequences”or “recombination sites” for that particular recombinase. Tyrosinerecombinases and serine recombinases are the two main families ofsite-specific recombinase. Examples of site-specific recombinase systemsinclude the Cre/Lox system (Cre recombinase mediates recombinationbetween loxP), the Flp/Frt system (Flp recombinase mediatesrecombination between FRT sites), and the PhiC31 system (PhiC31recombinase mediates DNA recombination at sequences known as attB andattP sites). Recombinase systems similar to Cre include the Dre-rox,VCre/VloxP, and SCre/SloxP systems (Anastassiadis K, et al. (2009) DisModel Mech 2(9-10):508-515; Suzuki E, Nakayama M (2011) Nucl. Acids Res.(2011) 39 (8): e49. It should be understood that reference to aparticular recombinase system is intended to encompass the variousengineered and mutant forms of the recombinases and recombination sitesand codon-optimized forms of the coding sequences known in the art. DNAplaced between two loxP sites is said to be “floxed”. A gene may bemodified by the insertion of two loxP sites that allow the excision ofthe floxed gene segment through Cre-mediated recombination. In someembodiments, expression of Cre may be under control of a cell typespecific, cell state specific, or inducible expression control element(e.g., cell type specific, cell state specific, or inducible promoter)or Cre activity may be regulated by a small molecule. For example, Cremay be fused to a ligand binding domain of a receptor (e.g., a steroidhormone receptor) so that its activity is regulated by receptor ligands.Cre-ER(T) or Cre-ER(T2) recombinases may be used, which comprise afusion protein between a mutated ligand binding domain of the humanestrogen receptor (ER) and Cre, the activity of which can be induced by,e.g., 4-hydroxy-tamoxifen. Placing Lox sequences appropriately allows avariety of genomic manipulations.

In some embodiments, step b) of the method comprises contacting the cellwith a protein synthesis inhibitor, solubilizing the cell plasmamembrane, and immunoprecipitating the ER.

The protein synthesis inhibitor is not limited and may be any suitableprotein synthesis inhibitor that keeps the labeled translocon associatedwith the ER. In some embodiments, the protein synthesis inhibitor blockstranslational elongation. In some embodiments, the protein synthesisinhibitor is one identified in Chan et. al., Eukaryotic proteinsynthesis inhibitors identified by comparison of cytotoxicity profiles,RNA 2004. 10: 528-543. In some embodiments, the protein synthesisinhibitor is cyclohexamide.

In some embodiments, the cell plasma membrane is solubilized withstep-wise concentrations of detergent. In some embodiments, the plasmamembrane followed by the ER membrane are solubilized in a step-wisemanner. Any suitable detergent or combinations of detergents known inthe art may be used and are not limited. Methods of solubilizing plasmamembrane can be practiced by those skilled in the art. In someembodiments, the detergent is digitonin and/or n-Dodecyl-B-D-Maltoside(DDM).

Methods of immunoprecipitation are also not limited and may be by anysuitable method known in the art. In some embodiments, the ER isimmunoprecipitated with an antibody specific for the label or for thelabeled translocon component. In some embodiments, the label is GFP andthe antibody is an anti-GFP antibody. In some embodiments, the antibodyis attached to a magnetic bead or other substrate.

The method of sequencing the mRNA is not limited and may be any suitablemethod known in the art. In some embodiments, the mRNA is sequenced bynext generation sequencing.

The cell of the methods and compositions described herein is not limitedand may be any suitable cell. In some embodiments, the cell is a stemcell (e.g., an embryonic stem cell, a mammalian embryonic stem cell, ahuman embryonic stem cell, a murine embryonic stem cell). In someembodiments, the cell is an embryonic stem cell. In some embodiments,the cell is an induced pluripotent stem cell.

In some embodiments, cells include somatic cells, stem cells, mitotic orpost-mitotic cells, neurons, fibroblasts, or zygotes. Stem cells mayinclude totipotent, pluripotent, multipotent, oligipotent and unipotentstem cells. Specific examples of stem cells include embryonic stemcells, fetal stem cells, adult stem cells, and induced pluripotent stemcells (iPSCs) (e.g., see U.S. Published Application Nos. 2010/0144031,2011/0076678, 2011/0088107, 2012/0028821 all of which are incorporatedherein by reference).

Somatic cells may be primary cells (non-immortalized cells), such asthose freshly isolated from an animal, or may be derived from a cellline capable of prolonged proliferation in culture (e.g., for longerthan 3 months) or indefinite proliferation (immortalized cells). Adultsomatic cells may be obtained from individuals, e.g., human subjects,and cultured according to standard cell culture protocols available tothose of ordinary skill in the art. Somatic cells of use in aspects ofthe invention include mammalian cells, such as, for example, humancells, non-human primate cells, or rodent (e.g., mouse, rat) cells. Theymay be obtained by well-known methods from various organs, e.g., skin,lung, pancreas, liver, stomach, intestine, heart, breast, reproductiveorgans, muscle, blood, bladder, kidney, urethra and other urinaryorgans, etc., generally from any organ or tissue containing live somaticcells. Mammalian somatic cells useful in various embodiments include,for example, fibroblasts, Sertoli cells, granulosa cells, neurons,pancreatic cells, epidermal cells, epithelial cells, endothelial cells,hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells,melanocytes, chondrocytes, lymphocytes (B and T lymphocytes),macrophages, monocytes, mononuclear cells, cardiac muscle cells,skeletal muscle cells, etc. In some embodiments, the cell is abeta-cell.

In some embodiments, the cell is a diseased cell or exhibits apathological state. In some embodiments, the cell is differentiated cellfrom an induced pluripotent stem cell. In some embodiments, the inducedpluripotent stem cell is derived from a subject having a disease orcondition of interest. In some embodiments, the induced pluripotent stemcell is from a subject having diabetes or a risk of developing diabetes.

The diseases and conditions are not limited. In some embodiments, thediseases or conditions are selected from a metabolic disease, acardiovascular disease, a circulatory or vascular disease, aneurological disease, a gastrointestinal disease (e.g., inflammatorybowel disease, Crohn's disease), and a disease associated with aging.

In some embodiments, the cell is undergoing a stress response (e.g.,hypoxia, hyperglycemia, hypoglycemia, hypoxia/reperfusion).

In some embodiments, the cell is responding to a stimulus when contactedwith a protein synthesis inhibitor. In some embodiments, the stimulus isa hormone (e.g., insulin). In some embodiments, the stimulus is anenvironmental condition (e.g., low oxygen, reperfusion). In someembodiments, the stimulus is insulin.

In some embodiments, the method further comprises performing the methodof enriching for mRNAs coding for secreted and membrane bound proteinson a control cell, and comparing the mRNA's isolated from the cell tothe mRNA's isolated from the control cell.

Applications of ER-Seq:

1. ER-seq can be used to compare in-vitro generated beta-cells fromnon-diabetic and diabetic patients to find novel disease biomarkers byspecifically isolating RNAs that code for secreted proteins andcomparing them among the two test groups.

2. ER-seq can be used to identify secreted peptide biomarkers producedby dysfunctional beta cells.

3. Induce stress response in vitro to identify biomarkers for beta-celldysfunction

4. By generating assays that look for the elimination of the secreteddistressed protein, we can find ways to protect beta-cells at the onsetof T1D.

5. Marking other cells and discovering their complement of secretedproteins.

Non-Human Animals

Some aspects of the invention are directed to a non-human animal capableof expressing a labeled SEC61b protein. In some embodiments, expressionof the labeled protein is inducible. In some embodiments, the labeledprotein has Cre-dependent expression. In some embodiments, the non-humananimal has inducible expression of the labeled SEC16b protein inbeta-cells. The label is not limited and may be any suitable label inthe art. In some embodiments, the label is a fluorescent protein. Insome embodiments, the label is Green Fluorescent Protein (GFP). In someembodiments, the non-human animal is a mouse or rat. In someembodiments, the non-human animal is a model of diabetes (e.g., NODmodel of type 1 diabetes, model of type 1 diabetes).

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane,D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, AManual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J.,2005. Non-limiting information regarding therapeutic agents and humandiseases is found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or11th edition (July 2009). Non-limiting information regarding genes andgenetic disorders is found in McKusick, V. A.: Mendelian Inheritance inMan. A Catalog of Human Genes and Genetic Disorders. Baltimore: JohnsHopkins University Press, 1998 (12th edition) or the more recent onlinedatabase: Online Mendelian Inheritance in Man, OMIM™ McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), as of May 1, 2010, ncbi.nlm.nih.gov/omim/ andin Online Mendelian Inheritance in Animals (OMIA), a database of genes,inherited disorders and traits in animal species (other than human andmouse), at omia.angis.org.au/contact.shtml. All patents, patentapplications, and other publications (e.g., scientific articles, books,websites, and databases) mentioned herein are incorporated by referencein their entirety. In case of a conflict between the specification andany of the incorporated references, the specification (including anyamendments thereof, which may be based on an incorporated reference),shall control. Standard art-accepted meanings of terms are used hereinunless indicated otherwise. Standard abbreviations for various terms areused herein.

Specific examples of certain aspects of the inventions disclosed hereinare set forth below in the Examples.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The details of thedescription and the examples herein are representative of certainembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Modifications therein and other uses will occurto those skilled in the art. These modifications are encompassed withinthe spirit of the invention. It will be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention provides all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. It is contemplated that all embodiments described herein areapplicable to all different aspects of the invention where appropriate.It is also contemplated that any of the embodiments or aspects can befreely combined with one or more other such embodiments or aspectswhenever appropriate. Where elements are presented as lists, e.g., inMarkush group or similar format, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. It should be understood that, in general, wherethe invention, or aspects of the invention, is/are referred to ascomprising particular elements, features, etc., certain embodiments ofthe invention or aspects of the invention consist, or consistessentially of, such elements, features, etc. For purposes of simplicitythose embodiments have not in every case been specifically set forth inso many words herein. It should also be understood that any embodimentor aspect of the invention can be explicitly excluded from the claims,regardless of whether the specific exclusion is recited in thespecification. For example, any one or more nucleic acids, polypeptides,cells, species or types of organism, disorders, subjects, orcombinations thereof, can be excluded.

Where the claims or description relate to a composition of matter, e.g.,a nucleic acid, polypeptide, cell, or non-human transgenic animal, it isto be understood that methods of making or using the composition ofmatter according to any of the methods disclosed herein, and methods ofusing the composition of matter for any of the purposes disclosed hereinare aspects of the invention, unless otherwise indicated or unless itwould be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise. Where the claims ordescription relate to a method, e.g., it is to be understood thatmethods of making compositions useful for performing the method, andproducts produced according to the method, are aspects of the invention,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. It is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the series, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is recited. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” generallyincludes numbers that fall within a range of 1% or in some embodimentswithin a range of 5% of a number or in some embodiments within a rangeof 10% of a number in either direction (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue). It should be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. It should also beunderstood that unless otherwise indicated or evident from the context,any product or composition described herein may be considered“isolated”.

EXAMPLES Example 1

Research Goal: To Find Novel Hormones to Cure Diabetes

A protocol to direct the differentiation of human embryonic or inducedpluripotent stem cells into functional, insulin expressing beta-cellswas developed previously¹. These Stem Cell-derived beta-cells (SC-beta)can be used to study the development of beta-cells, beta cell functionand physiology and they have the potential to treat diabetes by celltransplantation.

Hormones, including insulin, are secreted proteins with potent roles incontrolling metabolism, cellular differentiation, and disease. mRNAsencoding secreted or transmembrane proteins transit through the roughendoplasmic reticulum. To identify novel secreted and transmembraneproteins, a technique called Endoplasmic Reticulum Sequencing (ER-seq)has been developed that enriches RNAs of secreted/transmembrane proteinsby physically isolating actively translating ribosomes at the surface ofthe endoplasmic reticulum. To find novel hormones that regulate glucosemetabolism, the ER-seq method was applied to SC-beta cells and theassociated mRNA sequenced.

Next, differential gene expression and gene ontology analysis wereperformed to find all known secreted activities in SC-beta cells. Indoing so, the technology was validated by identifying, among many othersecreted proteins, the INSULIN gene. Eight genes without ascribedfunction or annotation were analyzed. Since these novel genes are madeby SC-beta cells and may code for secreted proteins, it was reasonedthat, like insulin, some of these novel genes might have a metabolicrole.

These eight genes were transiently expressed from plasmid DNA at highlevels in the liver of mice by hydrodynamic tail vein (HTV) injection ofplasmid DNA². To assess if any of these eight genes affected glucosehomoeostasis, three days after injection, at the time of high expressionfrom liver, a glucose tolerance test was performed.

One of the eight human genes injected, C1ORF127, cleared glucose fromthe blood circulation faster than controls (FIG. 1A). The reduction inglucose levels is significant and reproducible: to date, and wassuccessfully tested C1ORF127 glucose lowering activity in over fiftymice. Additionally, it was found that C1ORF127 is able to reduce bloodglucose levels in diet-induced obese mice, a model for Type2 diabetes.

Since C1ORF127 is expressed in INSULIN producing beta-cells, it wasasked if its glucose lowering activity was dependent on insulin action.To test this point, a potent and specific peptide inhibitor, andantagonist of the insulin receptor, S9613 was used. When S961 isadministered acutely to mice they quickly become hyperglycemicdemonstrating the efficacy of S961 at inhibiting the insulin receptorand preventing glucose uptake into muscle and fat, thereby resulting ina net accumulation of glucose in the circulation. HTV injections ofC1ORF127 and control DNA and on day three performed a glucose tolerancetest. For this experiment, S961 was added at two timepoints; first, twohours before the injection of glucose; and second, with the glucosebolus at the beginning of the test. As seen in FIG. 1B, C1ORF127 canclear glucose from the circulation faster than controls. This datastrongly suggests that C1ORF127 removes glucose from the circulationindependent of insulin action. Notably, in these experiments, C1ORF127lowers blood glucose levels without causing hypoglycemia, a problem withall known drugs that work to promote INSULIN secretion (Time 0 min inFIG. 1A and D3 fasted in FIG. 1B).

The C1ORF127 gene is predicted to code for a protein of 684 amino acids(MW 73 kDa). It is a highly conserved transcript among vertebrates thatlacks canonical signals for secretion or membrane insertion. Itsexpression has been confirmed in SC-beta cells and cadaveric humanIslets by immunofluorescence microscopy. Using internal expressiondatabases, C1ORF127 was found to be expressed primarily in beta-cellsand at lower levels in somatostatin-expressing (pancreatic delta) cells.In humans, C1ORF127 is also expressed in muscle and cerebellum. Itsmouse orthologue, Gm572, is expressed exclusively in beta- and delta-(somatostatin) expressing cells. By Western blot, the protein is foundto be expressed at the predicted molecular weight in SC-beta andcadaveric human islets. Moreover, the Type 2 Diabetes knowledge portal(a curated disease risk repository) was search and identified mutationsin C1ORF127 that might be critical for the development or progression ofType 2 diabetes⁴.

REFERENCES

-   1. Pagliuca F W and Millman J R, et. al., Generation of functional    human pancreatic β cells in vitro. Cell. 2014 Oct. 9; 159(2):428-39-   2. Chen C A, et. al., In vivo screening for secreted proteins that    modulate glucose handling identifies interleukin-6 family members as    potent hypoglycemic agents. PLoS One. 2012; 7(9): e44600.-   3. Schaffer L, et. al., A novel high-affinity peptide antagonist to    the insulin receptor. Biochem Biophys Res Commun. 2008 Nov. 14;    376(2):380-3.-   4. Type 2 Diabetes Knowledge Portal. 2018 Sep. 10    www.type2diabetesgenetics.org/home/portalHome

Example 2

The biogenesis of hormones is directed by endoplasmic reticulum(ER)-localized ribosomes actively translating their mRNAs at thetranslocon complex (Mandon et al., 2013; Ogg et al., 1995; Rapoport etal., 2007). Most studies identifying novel secreted factors and hormoneshave relied on algorithms that predict canonical topogenic signals(Diehn et al., 2000; Emanuelsson et al., 2007; Kall et al., 2004;Meinken et al., 2015, Petersen et al., 2011). Although informative,these computational predictions do not efficiently account for asignificant fraction of genes that are part of this functional class(Jan et al., 2014). Recent efforts to characterize the complement ofmRNAs of secreted factors relied on the biochemical isolation ofER-localized ribosomes (Jan et al., 2014; Fazl et al., 2019; Reid etal., 2014). However, these efforts have been limited to yeast and cancercells in vitro (Jan et al., 2014; Fazl et al., 2019; Reid et al., 2014).Here is described a protocol called ER-seq for the biochemical isolationof ribosome/translocon complexes in human pluripotent stem cells (hPSCs)and differentiated progeny. This protocol was applied to SC-β cells andidentified a previously uncharacterized gene, C1ORF127, that promotesglucose clearance independent of insulin action.

Development of a Biochemical Fractionation Method for the Isolation ofRibosome-Translocon Complexes

As the biogenesis of secreted factors and hormones occurs inER-localized ribosome/translocon complexes, it was reasoned that theisolation of translocon-associated mRNAs will effectively enrich formRNAs of secreted factors and may serve as a proxy for their expressionin a cell (Jan et al., 2014). To isolate translocon complexes, hPSC celllines were generated that express a subunit of the translocon complex,Sec61β, fused to GFP either constitutively or in insulin-expressing βcells (FIG. 2A). To achieve ubiquitous and constitutive expression inhPCSs and differentiated progeny, TALEN-mediated genome editingtechnology was used to knock-in the GFP-SEC610 fusion protein into theAAVS1 locus under the control of a ubiquitously-expressedartificially-engineered CAAGS promoter (CAAGS::GFP-SEC61β; FIG. 2B).Similarly, to express this transgene in insulin-expressing β cells,CRISPR was used to knock-in the GFP-SEC61β fusion transgene into thelast exon of the endogenous insulin gene (INS::GFP-SEC61β; FIG. 2C). InhPSCs and SC-β cells, the expression of the transgene was perinuclear asexpected for an ER-localized protein (FIGS. 2B-2C).

Protocols that rely on the differential solubility of cellular membranesto permeabilize the plasma and ER membranes in a step-wise manner weredeveloped (FIG. 4.2A). First, digitonin was used to permeabilize theplasma membrane and retrieve the cytoplasmic fraction. To the insolublefraction, n-Dodecyl-B-D-Maltoside (DDM) was added in a hypotonic bufferto permeabilize the ER membrane and luminal components which was calledthe ER fraction (Nichitta et al. 2014). After ER permeabilization, theER fraction was subjected to immunoprecipation with anti-GFP magneticbeads to purify ribosome/translocon complexes and associated mRNAs (FIG.3A). This protocol was applied to self-renewing hPCSs and detected asignificant enrichment in Sec61β and GFP protein expression in theimmunopurified (IP) fraction relative to unfractionated cell extracts asassayed by western blot (FIG. 3B). Importantly, the ribosomal proteinsubunit L13a and ribosomal RNA subunits 28S and 18S were alsoco-purified (FIGS. 3B-C). The immunopurified ER fraction was subjectedto mass spectrometry and detected peptides of the translocon subunitSEC61a, translocon-associated protein disulfide isomerase (PDI) andmultiple ribosomal protein subunits (FIG. 3D). In all, the biochemicalprotocol allows for a robust and effective enrichment ofribosome/translocon complexes.

ER-Seq Robustly Enriches for mRNAs of Secreted Factors in hPSCs and SC-βCells.

To determine whether this approach effectively enriches for mRNAs thatcode for secreted factors and membrane proteins, microarray analysis ontranslocon-associated mRNAs purified from hPSCs was performed. Comparedto mRNAs collected from total unfractionated cell extracts, anenrichment of mRNAs encoding for the ER factors Sec61α and DDOST as wellas secreted factors such as BMP7, DLL1, COL2A1, COL7A1, among others,was detected (FIG. 4A). Out of 3174 genes that are enriched in the IPfraction relative to total mRNA, 989 genes (31.1%) were detected thatare predicted to be secreted factors or membrane proteins based oncanonical topogenic signal peptide prediction (FIG. 4B). Based on geneontology analysis of genes enriched in the IP fraction, there is asignificant enrichment of genes that are part of the endomembranesystem, vesicles and extracellular components in the IP fraction (FIG.4C). 650 genes were also detected that are enriched in the IP fractionthat are unannotated and have no predicted localization signal. Asaround 10% of all genes expressed in most cell types are predicted to besecreted (Uhlen et al., 2015), computational analysis suggests thisapproach effectively enriches for mRNAs of secreted factors expressed inhPSCs.

The efficacy of the ER-seq protocol at enriching for mRNAs coding forsecreted factors in highly secretory β cells was next determined. Tothis end, an in vitro directed differentiation protocol (Pagliuca etal., 2014) to generate SC-β cells from hPSCs using the INS::GFP-SEC61βcell line (FIG. 5A-B) was used. This allowed for isolation ofribosome/translocon complexes from β cells in a heterogeneous mixture ofcell types and RNA-sequencing on translocon-associated mRNAs. Theprotocol was applied to SC-β cells and, by RNA-sequencing oftranslocon-associated mRNAs, identified a significant enrichment ofhormones such as insulin and amylin (IAPP), angiogenic factors VEGFA andVGF, as well as other genes involved in insulin secretion such aschromogranin A (CHGA), secretogranins (SCG2, SCG3, SCG5), andsynapthophysin (SYP) (FIG. 5C). 2,732 genes were identified that areenriched in the IP fraction relative to total unfractionated RNA (foldchange>2). 874 of this set of genes (32%) are predicted to be secretedfactors or membrane proteins based on computational topogenic signalprediction (Kall et al., 2005) (FIG. 5D). 17% of the IP-enriched genesdid not have a predicted subcellular localization pattern and were notannotated as nuclear, cytoplasmic, secreted or membrane-localized. Amongfactors predicted to be part of the secretome of the cell, a robustenrichment of their mRNAs in the IP fraction relative to total RNA (FIG.5E) was detected. Genes that are annotated as nuclear and/or cytoplasmicwere depleted in the IP fraction (FIG. 5E). Accordingly, gene ontologyanalysis of IP-enriched genes suggests an enrichment in factors that arepart of the endomembrane system, ER and extracellular part of the cells(FIG. 5F). Overall, computational analysis suggests an effectiveenrichment and quantification of mRNAs of secreted factors by ER-seq.

Stage-Specific Expression Patterns of Translocon-Associated mRNAs

Analysis of translocon-associated mRNAs in SC-β cells revealed anenrichment of a significant number of genes that may represent novelsecreted factors expressed in SC-β cells. To identifytranslocon-associates genes that are preferentially expressed in pcells, the ER-seq protocol was applied to cells at multiple stages ofthe in vitro differentiation of β cells. To do that, the constitutiveexpression of GFP-SEC61β in the CAAGS::GFP-SEC61β cell line was reliedupon to isolate translocon-associated mRNAs in hPSCs and theirdifferentiated progeny during the early stages of differentiation (FIG.6A). During the last stages of differentiation, the INS::GFP-SEC61β cellline was used to isolate translocon-associated mRNAs ininsulin-expressing SC-β cells (FIG. 6A). Translocon-associated mRNAs wassequenced at all stages of differentiation and stage-specific geneexpression signatures were identified (FIG. 6B). 601 genes that aredifferentially expressed across all stages of differentiation wereidentified. A gene expression signature was found that was specific toSC-β cells that include 139 genes, 44 of which are predicted secretedfactors. Gene ontology analysis of SC-β cell-enriched genes showed asignificant enrichment of factors involved in insulin secretion, glucosehomeostasis, extracellular space, secretory granules, as well as othercategories that correlate with secretion and membrane-targetingprocesses (FIG. 6C-D). Genes involved in insulin secretion and that arepreferentially expressed in endocrine cells are significantlyupregulated in SC-β cells compared to earlier stages of differentiation(FIG. 6E). Interestingly, 11 unannotated genes that also display anexpression pattern specific to SC-β cells (FIG. 6F) were identified. Inall, this analysis decodes gene expression signatures that correlatewith the differentiation of SC-β cells in vitro and identified a set ofgenes that are preferentially expressed in this cell type.

Example 3

GreenER Reporter Mouse

A mouse Cre-dependent reporter transgene with the AcGFP-SEC61b fusionprotein described above (ROSA-floxed-STOP-floxed-AcGFP-SEC61b) isdeveloped herein and called the GreenER reporter. This mouse has beencrossed to Insulin-Cre mice to generate B-cells whose endoplasmicreticulum fluoresces green, enabling the application of the ER-seqtechnology described above. This B-cell specific GreenER strain can becrossed into the NOD model of Type 1 diabetes, giving researchers theability of finding secreted biomarkers during the course of disease.

Additionally, since some of the stressors common to Type 1 and Type 2diabetes may be similar, this strain maybe crossed into models of Type 2diabetes such as the ob/ob and the db/db models.

For mouse ER-seq, the genetic component is the targeting of GreenFluorescent Protein (GFP) fused to an integral component of the ERtranslocon, Sec61b, to the ROSA locus. Here, a GFP-Sec61b fusion proteinwas located behind a floxed-flanked transcriptional stop signal. Hence,only in the presence of CRE-recombinase, the transcriptional stopcassette is removed and GFP-Sec61b is expressed marking the ER ofCRE-expressing cells with green fluorescence. The biochemical componentinvolves the development of methods to perform subcellular fractionationto make ER-microsomes that preserve the mRNA-ribosome-transloconinteraction. Immunoprecipitation using antibodies specific to GFP toprecipitate this complex were then performed. The molecular biologycomponent relies on extracting the translocon associated mRNA's from thecomplex and in performing transcriptional analysis of these mRNA's viaRNA sequencing.

FIG. 25A shows the targeting vector and a positively targeted mouseembryonic stem cell (mESC) colony that was infected with CRE virus toshow that the transgene can mark the ER with high fidelity. This colonywas used to make our founder reporter mouse strain namedRosa-floxed-STOP-floxed:: Green-ER (“Green-ER” Reporter).

FIG. 25B shows the in vivo validation of the reporter strategy. TheGreen-ER reporter mouse strain was crossed with a ubiquitous CREexpressing mouse (CMV-Cre). Tail tip fibroblasts were generated fromCMV-CRE/Green-ER progeny (Ubiquitous Green-ER mouse) and is shown thatthe GFP signal in all cells is specific to the ER (false colored yellowin this image).

FIG. 25C shows pancreatic sections from progeny of crosses between theGreen-ER reporter mouse strain to a beta-cell CRE expressing mouse(Ins2-Cre). This strain was named the beta-cell Green-ER mouse. Therecombination is restricted to islet beta-cells and the reporterexpression is specific to the ER.

Example 4—Methods

Generation of Transgenic Cell Lines

All the experiments were performed using the human embryonic stem cellline HUES8 obtained from the Human Embryonic Stem Cell Facility and iPSCore Facility of the Harvard Stem Cell Institute. gRNA sequences for theinsulin genomic region were ligated into either eCas9 (Addgene 71814) orLbCpf1 (Addgene 84742) CRISPR plasmids. Homology arms flanking ˜750 bpupstream and downstream of the stop codon in the last exon of theinsulin gene were generated by PCR with primers flanking this region. 5′and 3′ homology arms were ligated were ligated to 2A-Sec61β-GFPtransgene and a puromycin antibiotic selection marker. For thegeneration of CAAGS:: Sec610-GFP cell line, TALEN constructs weredesigned to target the safe-harbor AAVS1 locus. 5′ and 3′ homology armswere ligated were ligated to 2A-PURO (puromycing resistance gene), alinker and CAAGS promoter driving the expression of the Sec61β-GFPtransgene. HUES8 cells were dispersed into single cells using TrypLEExpress and transfected with targeting vectors using the Nucleofectorkit (Invitrogen). 72 hr post-electroporation cells were treated withpuromycin at a concentration of 1 g/mL for 7 days to obtain singlecolonies. Colonies were picked under a microscope around 18-21 days postelectroporation into a 96 well plate and expanded. Genomic DNA (gDNA)from the 96 well plate was extracted using the Zymo Research Quick-DNA96 Plus Kit and insertion of 5′ and 3′ homology arms was confirmed withPCR. Clones that were confirmed as karyotypically normal by karyotypeanalysis through Cell Line Genetics were used for directeddifferentiation towards beta cells.

Differentiation of SC-β Cells

Human pluripotent stem cells (hPSCs) were maintained in mTeSR1 (StemCell Technologies) in 500 mL spinner flaks on a stir plate (Chemglass)set to 70 rpm in a 37° C. incubator, 5% C02, and 100% humidity.Differentiations into SC-β cells were performed following a protocoldescribed previously (Pagliuca et al., 2014) as follows: HUES8 cellswere seeded at 6×10⁵ cells/mL in mTeSR1 media and 10 μm Y27632(Sigma-Aldrich). The media was changed 48 h later and thedifferentiations were started 72 h after the cells were seeded. Themedia changes were as follows:

Stage 1 definitive endoderm: S1+100 ng/mL ActivinA (R&D Systems)+3 μMChir99021 (Stemgent) in day 1 and S1+100 ng/mL ActivinA on day 2.

Stage 2 gut tube endoderm: days 4, 6: S2+50 ng/mL KGF (Peprotech).

Stage 3 pancreatic progenitor 1: days 7, 8: S3+50 ng/mL KGF+0.25 μMSant1 (Sigma)+2 μM RA (Sigma)+200 nM LDN193189 (only Day 7) (Sigma)+500nM PdBU (EMD Millipore).

Stage 4 pancreatic progenitor 2: days 9, 11, 13: S3+50 ng/mL KGF+0.25 μMSant1+100 nM RA+10 μm Y27632+5 ng/mL Activin A.

Stage 5 endocrine progenitors: Days 14, 16: S5+0.25 μM Sant1+100 nM RA+1μM XXI (EMD Millipore)+10 μM Alk5i II (Axxora)+1 μM T3 (EMDMillipore)+20 ng/mL Betacellulin (Thermo Fisher Scientific). Days 18,20: S5+25 nM RA+1 μM XXI+10 μM Alk5i II+1 μM T3+20 ng/mL Betacellulin.

Stage 6 β cells: S3 media change every other day. In the final stage,cells were analyzed between 7 and 21 after stage 6 differentiation wasstarted.

Biochemical Fractionation Protocol

Around 100×10⁶ cells were collected from differentiation spinner flasks.To stall ribosomes, 100 μg/mL cycloheximide (CHX) was added andincubated for 10 mins. All the following steps of the fractionation wereperformed with solutions containing 100 μg/mL cycloheximide(Sigma-Aldrich). Cell clusters were washed in PBS/CHX. 4 mL ofAccutase/CHX was added to suspension of cell clusters and incubated for7 mins RT. Clusters were then dissociated by mechanical dissociationusing pipettes, resuspended in PBS/CHX and cells were pelleted withcentrifugation for 5 mins at 230 rcf at RT. Pellet was resuspended in 3mL of cytoplasmic buffer containing 110 mM potassium acetate(Sigma-Aldrich), 25 mM K-HEPES (Sigma-Aldrich), 15 mM magnesium chloride(Sigma-Aldrich), 4 mM calcium chloride (Sigma-Aldrich), 0.015%digitonin, 1.0 mM dithiothreitol (Sigma-Aldrich), 100 μg/ml CHX, 1×cOmplete protease inhibitor cocktail (Millipore) and 500 U/ml RNasinribonuclease inhibitors (Promega) and incubated for 20 mins at 4° C.Cell suspension was centrifuged for 5 mins at 845 rcf at 4° C. andsupernatant (cytoplasmic fraction) and stored at −80° C. for subsequentanalysis. The pellet was resuspended in 1 mL ER permeabilizationhypotonic buffer containing 20 mM HEPES, 1.5 mM MgCl₂, 0.42M NaCl, 0.2mM EDTA, 25% glycerol, 2% n-Dodecyl j-D-maltoside (DDM), 1.0 mM DTT, 100μg/ml CHX and 1× cOmplete protease inhibitor cocktail (Millipore) and500 U/ml RNasin ribonuclease inhibitors (Promega) and 50 uL magneticagarose binding control beads (Chromotek) and incubated for 1 hour at 4°C. rotating head-to-tail. GFP-MA-TRAP beads (Chromotek) were blocked in1% BSA-PBS solution for at least 30 mins on ice. Cell extract washomogenized using a Wheaton Glass Homogenizer by slowly moving pestle upand down 15 times. The extract was centrifuged for 5 min at 850 rcf at4° C. Tube with blocked GFP-MA-TRAP beads was inserted into a DynaMag-2magnetic stand (Thermo Fisher Scientific) and the solution wasdiscarded. Supernatant was then added to GFP-MA-TRAP bead slurry andincubated for 30 mins at 4° C. with head to tail rotation. Samples wereinserted into magnetic stand to collect the unbound fraction and beadswere subsequently washed with 500 μL ER Permeabilization buffer twice.After the washes, 500 ul of Trizol was added to beads and stored at −80°C.

RNA Extraction, Library Preparation and Sequencing

Subcellular fractions stored in TRIzol reagent were combined with 0.2 mLchloroform per 1 mL TRIZol reagent used and incubated for 2-3 minsfollowed by centrifugation for 15 mins at 12,000 rcf at 4° C. Theaqueous phase containing the RNA was combined with 0.5 mL isopropanolper 1 mL TRIZol reagent used. After incubating for 10 mins on ice, RNAwas precipitated for 10 minutes at 12,000×g at 4° C. Pellet was washedin 1 mL 75% ethanol and air dried for 5-10 mins. RNA was stored at −80°C. in RNA storage solution (Invitrogen) for subsequent analysis.

Precipitated RNA was subjected to in vitro RNA amplification withreverse transcription following a published protocol (Hashimshony etal., 2012). Reverse-transcription primers were designed with an anchoredpolyT, the 5′ Illumina adaptor of Illumina small RNA kit and a T7promoter. The MessageAmp II RNA kit (Ambion) was used with the modifiedreverse-transcription primer (Hashimshony et al., 2012). The reactionwas performed with 50 ng of RNA and 25 ng/uL amplification primersfollowing MessageAmp II RNA kit's protocol. cDNA cleanup was performedwith AMPure XP beads (Beckman coulter) by magnetic bead isolation,cleanup with 80% EtOH followed by in vitro transcription for 13 hoursfollowing MessageAmp II RNA kit's protocol. RNA was fragmented in 200 mMTris-acetate [pH 8.1], 500 mM KOAc, 150 mM MgOAc and reaction wasstopped by placing on ice and the addition of one-tenth the volume of0.5M EDTA, followed by RNA cleanup. RNA quality and yield were assayedusing a Bioanalyzer (Agilent). Illumina's directional RNA sequencingprotocol was used and only the 3′ Illumina adaptor was ligated. A totalof 12 cycles of PCR with elongation time of 30 s was performed.Libraries were sequenced using the NextSeq 500 platform (Illumina)according to standard protocols. Paired-end sequencing was performed,reading at least 15 bases for read 1, and 50 bases for read 2, andIllumina barcodes.

Transcriptomic Analysis

Reads were trimmed for universal ilumina adapters and polyA sequenceswith Cutadapt v1.8.1 and assessed for quality control using Fastqcv0.11.5. Reads were aligned to the human reference genome (hg38) withTophat v2.1.1 using default parameters. Downstream transcriptquantification and differential gene expression analysis was performedwith Cufflinks v2.2.1 (Trapnell et al., 2013). Differentially expressedgenes were defined as those with adjusted p-values below 0.05 using thecuffdiff algorithm.

Gene Ontology Analysis

Differentially expressed genes (adjusted p-value<0.05) were used forgene ontology analysis using WebGestalt GSAT (Wang et al., 2017) andingenuity pathway analysis (IPA, QIAGEN Inc.,www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis)). Geneontology terms and enriched pathways with P-value<0.05 were consideredas enriched in differentially expressed genes.

Prediction of Secreted Factors

Translated protein sequences from candidate transcripts were submittedto the Phobius algorithm to predict signal peptide using a hidden Markovmodel (Kall et al., 2005). Transcripts with predicted signal peptide butno predicted transmembrane topology were classified as part of thesecretome of the cell. Ingenuity pathway analysis (IPA, QIAGEN Inc.,www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) wasalso used to classify candidate transcripts as cytoplasmic or nuclear.

Western Blot Analysis

After removing the aqueous phase for RNA extraction as described above,the phenol-ethanol was resuspended in 1.5 mL isopropanol per 1 mL TRIzolreagent used and incubated for 10 mins. After centrifugation for 10 minsat 12,000 g at 4° C., the pellet was washed in 2 mL of 0.3 M guanidinehydrochloride in 95% ethanol per 1 mL TRIzol reagent used, incubated for20 min at RT followed by centrifugation for 5 mins at 7500 g at 4° C.This step was repeated twice. In the final, 2 mL of 100% ethanol per 1mL TRIzol used was added and incubated for 20 mins. After centrifugationfor 5 mins at 7500 g at 4° C., pellet was air dried for 5-10 mins andresuspended in 200 uL of 1% SDS buffer. Protein concentration wasmeasured using the BCA Protein Assay kit (Thermo Scientific). 5-10 ug ofprotein extracts were separated by AnyKD Mini-Protein precast gels(Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad).Membranes were blocked in 3% BSA+0.1% Tween 20 TBS for 30 mins at RT andthen incubated with the following primary antibodies overnight at 4° C.:mouse anti-Sec61β (Santa Cruz, sc-393633), chick anti-GFP (Aves,GFP1020), rabbit anti-ribosomal protein L13a (Cell signaling, 2765).After washing, the membranes were incubated with HRP-conjugatedsecondary antibodies for 1 h at RT, and then incubated inchemiluminescent ECL detection reagent (VWR) for signal detection anddevelopment.

4.5.9 Animals Studies

All animal treatment, husbandry, procedures were done in accordance withinstitutional animal care standards. All protocols were approved byHarvard University's Institutional Animal Care and Use Committee.Initial screening of ER-seq candidates by tail vein injection wasperformed using ICR mice obtained from Jackson Laboratory. Additionaltail vein injections for characterization of c1orf127, including S961experiments, were performed using C57BL/6 mice from Charles RiverLaboratory. Streptasatozin induced diabetic mice were C57BL/6 miceobtained from Jackson Laboratory, STZ injections were performed byJackson Laboratory.

Candidate Cloning

Expression plasmids for screened ER-Seq candidates were obtained firstby PCR amplification from cDNA libraries of stem cell derivedbeta-cells; primers were designed to add gateway cloning sites. Becauseof difficulty amplifying C1orf127, a clone of the ORF was purchased fromDharmacon and amplified to add gateway sites. PCR amplicons were addedto PDonor gateway vector. The ORFs were then transferred into CaG highexpression vectors (for tail vein injection) and into lentiviralproduction vectors (cell line production). In addition to ER-Seqcandidates, controls including cytoplasmic GFP, nuclear TD-Tomato,furin-cleavable insulin, and a nanoluciferase were purchased as customgenes with gateways sites from Integrated DNA Technologies. Controlswere then cloned into the same vectors.

Tail Vein Injections

Mice were first anaesthetized by intraperitoneal injection 23 ul/gbodyweight of a 1.25% Avertin solution. Tails of anaesthetized mice werewarmed under a heat lamp to dilate the tail vein. Anaesthetized micereceived an injection of 80 ul/g bodyweight of saline with 35 ug ofexpression vector DNA in the tail vein over approximately 7 seconds. Forexperiments involving tail vein injected mice, mice were tested 48-72hrs after injections.

Standard Glucose Tolerance Tests

Mice were fasted overnight (5 pm to 9 am). Blood glucose measurementswere taken before fasting and immediately before glucose injections.Mice received an intraperitoneal injection of glucose at 2 g/kgbodyweight. Blood glucose measurements were then taken at 15, 30, 45, 60and 90 minutes post injection. Occasionally a 120 minute time point wascollected if blood glucoses had not yet fallen below normal levels (<250mg/dl).

S961 Glucose Tolerance Tests

Mice were fasted overnight (5 pm to 9 am). Blood glucose measurementswere taken before fasting, immediately before initial S961 injection andbefore glucose injection. Mice received an intraperitoneal injection of10 ul/g bodyweight of 45 uM S961. 2 hours after this initial injection,mice received an intraperitoneal injection of 10 ul/g bodyweight of 30uM S961, 15% D-Glucose (1.5 g/kg glucose). Blood glucose measurementswere then taken at 15, 30, 45, 60, 90, 120 minutes post injection, andthen measures were taken hourly until values fell below normal (<250mg/dl).

STZ Glucose Tolerance Tests

Blood glucose measurements were taken before tail vein injection, beforefasting and immediately before glucose injection. Only mice withinitially severe hyperglycemia (>400 mg/dl) were used. Mice were fastedovernight (5 pm to 9 am). Mice received an intraperitoneal injection of1.5 g/kg glucose. Blood glucose measurements were then taken at 15, 30,45, 60, 90, 120 minutes post injection, and then measures were takenhourly until values fell below normal (<250 mg/dl).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism software.Glucose tolerance comparisons were made using t-tests for bothindividual time-points. ANOVA and Tukey's multiple comparison tests wereused to compare areas under the curve.

REFERENCES

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Example 5

Alternative Sample ER-Seq Protocol

Permeabilization Buffer Stock (to Make Cytoplasmic Extraction Buffer)

for 50 ml 110 mM KOAc 1.83 ml of 3M 25 mM K-HEPES 7.2 2.5 ml of 0.5M 15mM MgCl2 750 μl of 1M 4 mM CaCl2 200 μl of 1M RNAse free water 44.72 ml

Hypotonic Buffer Stock (50 mL; to Make ER Permeabilization Buffer)

Nuclease-free water 32.2 mL 20 mM HEPES 1 mL of 1M 1.5 mM MgCl2 75 uL of1M 0.42M NaCl 4.2 mL of 5M 0.2 mM EDTA 20 uL of 0.5M 25% (v/v) glycerol12.5 mL of 100%

Cytoplasmic Buffer (2 mL)

Permeabilization Stock 951.5 μl × 2 0.015% Digitonin 30 μl of 1% 1.0 mMDTT 20 μl of 100 mM 100 μg/ml CHX 2.0 μl of 100 mg/ml 1X Protease InhCocktail 20.0 μl of 10X 500U/ml RNAsin 25.0 μl of 40U/p1

ER Permeabilization Buffer (4 mL)

Hypotonic Buffer Stock 3.066 mL 2% DDM 800 uL of 10% 1.0 mM DTT 40 μl of100 mM 100 μg/ml CHX 4.0 μl of 100 mg/ml Protease Inh Cocktail 40.0 μlof 10X 500U/ml RNAsin 50.0 μl of 40U/μl

CHX/PBS:100 μg/ml CHX (30 μl of 100 mg/ml CHX in 30 ml PBS w/o cations)

CHX/Accutase (8 ml): 8 μl CHX into 4 ml PBS+4 mL Accutase

GFP-MA TRAP magnetic beads (2×): 100 uL beads+100 uL BSA+400 uLpermeabilization stock (we have used 50 uL beads for 60×10⁶ cells)

10× Protease Inhibitor cocktail: 1 tablet in 1 ml DMSO

Modifications to the Protocol:

Collect 130 mL from 2 St5d7 spinner flasks (3×50 mL tubes)

Let clusters settle for 5 mins (remove all but 10 mL)

Add 10 uL CHX (100 ug/mL)

Incubate in rocker for 5 mins

Let clusters settle for 5 mins in 10 mL PBS/CHX

Add 4 mL CHX/Accutase and incubate for 7 mins gently shaking every 2mins.

Centrifuge for 5 mins @ 200 rcf

Resuspend in 3 mL cytoplasmic buffer, split into 2 1.5 mL tubes, andincubate for 20 mins

Centrifuge for 5 mins @ 845 g in cold room's centrifuge.

Add 1 mL ER permeabilization buffer and 50 uL GFP binding control beadsto each sample

Incubate for 1 hr and proceed to permeabilize and pulldown and describedbefore Homogenize using Wheaton Glass Homogenizer (moving the pestle upand down 15 times slowly)

Transfer extract to a new 1.5 mL centrifuge tube and spin down for 5 minat 3000 rpm in the cold room's centrigue

Add supernatant to pre-blocked GFP-MA-TRAP bead slurry (pellet is thenuclear fraction).

Incubate bead slurry with head to tail rotation in the cold room for 30min.

Retrieve tube from cold room and apply magnet for at least 1 min. Yourbound fraction should contain the translocon associated mRNAs. Collectthe supernatant into a 15 ml conical tube. This is your translocondepleted membrane fraction. Save 2 μl from the translocon depletedmembrane fraction for epifluorescence analysis. Add 2.5 ml of Trizol andfreeze at −80 C.

Wash beads twice with 500 μl ER Permeabilization buffer. Transfer to anew tube. Save 2 μl from your Translocon enriched fraction forepifluorescence analysis. Apply magnet and discard supernatant. Add 500ul of Trizol to beads. Vortex and freeze at −80° C.

1.-97. (canceled)
 98. A method of treating or preventing a disorderassociated with elevated blood glucose levels in a subject, comprisingadministering to said subject an effective amount of an agent thatincreases the level or activity of a C1ORF127 gene product.
 99. Themethod of claim 98, wherein the agent increases the level or activity,expression, or secretion of an endogenous C1ORF127 gene product whenadministered to the subject.
 100. The method of claim 98, whereinadministration of the agent corrects a genetic defect in the subjectcausing aberrant expression or activity of the C1ORF127 gene product.101. An agent that increases the level or activity of a C1ORF127 geneproduct when administered to the subject.
 102. The agent of claim 101,wherein the agent increases the level or activity, expression, orsecretion of an endogenous C1ORF127 gene product when administered tothe subject.
 103. The agent of claim 101, wherein the agent corrects agenetic defect in the subject causing aberrant expression or activity ofthe C1ORF127 gene product when administered to the subject.